Aldoxime dehydratase mutant and application thereof in catalytic synthesis of dinitrile compound

Abstract

The invention discloses an aldoxime dehydratase mutant and application of the aldoxime dehydratase mutant in catalytic synthesis of a dinitrile compound, which is characterized in that wild aldoxime dehydratase of SEQ ID NO: 2 is subjected to M29A and L318F double mutation and marked as TU-1. The mutant can efficiently catalyze oxidation of 5-hydroxymethylfurfural (HMF) to generate 2, 5-furandicarboxaldehyde (DFF), the DFF is further converted into 2, 5-furandicarboxaldehyde dioxime (DFFD) and a final target product 2, 5-dicyanofuran (DCF) under mild conditions, the reaction selectivity is high, and the yield is excellent. The invention provides a green and controllable technical scheme with remarkable industrial potential for preparing high value-added furan chemicals from renewable resources.

Description

An aldoxime dehydratase mutant and its application in the catalytic synthesis of dinitrile compounds Technical Field

[0001] This invention relates to the field of biocatalysis research, specifically to an aldoxime dehydratase mutant and its application in the catalytic synthesis of dinitrile compounds. Background Technology

[0002] Synthesizing chemicals from non-renewable resources such as fossil fuels not only faces the predicament of resource depletion but also poses environmental pollution risks. In recent years, renewable biomass has emerged as a "green raw material" for sustainable green development in chemical production and an effective way to address humanity's over-reliance on fossil fuels. C5 and C6 monosaccharides extracted from non-food biomass such as cellulose can be efficiently dehydrated to produce furfural and 5-hydroxymethylfurfural (HMF), which can then be used to synthesize furan-based fine chemicals, such as its derivative 2,5-dicyanfuran (DCF). 2,5-Dicyanfuran is a promising chemical; after hydrogenation and ring-opening, it can serve as an readily available raw material for synthesizing adiponitrile and other fine chemicals. Adiponitrile, a linear aliphatic dinitrile, is used in the production of nylon-66. Furthermore, dinitrile compounds, possessing two cyano functional groups, are key intermediates in organic chemical synthesis.

[0003] Currently, researchers have reported chemical synthesis methods for 2,5-dicyanfuran. For example, Xu's team reported a two-step reaction strategy to synthesize 2,5-dicyanfuran from 2,5-diformylfuran (DFF) using the solid acid catalyst Amberlyst-15, specifically involving oxime and dehydration (ACS Sustainable Chem. Eng. 2018, 6, 3, 2888–2892; CN109776462A). This method uses a reaction temperature of 120 °C and acetonitrile as the solvent. These methods not only use expensive transition metal ligands as catalysts but also involve harsh reaction conditions and the use of large amounts of highly toxic reagents, severely limiting the large-scale synthesis of dicyandiamide compounds.

[0004] Therefore, developing a simple and green synthetic strategy for 2,5-dicyanfuran and its analogues is of great significance. Summary of the Invention

[0005] The purpose of this invention is to address the shortcomings of existing technologies for the synthesis of 2,5-dicyanfuran and to provide an aldoxime dehydratase mutant, OxdF1-TU1, with high catalytic activity against furan substrates. This mutant can be used to catalyze the synthesis of high-value-added 2,5-dicyanfuran from 2,5-furandiformaldehyde dioxime (DFFD) water.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] In a first aspect, the present invention provides an aldoxime dehydratase mutant with a two-site mutation, specifically, the 29th methionine M of the wild-type aldoxime dehydratase of SEQ ID NO:2 is mutated to alanine A (M29A) and the 318th leucine L is mutated to phenylalanine F (L318F), denoted as TU-1.

[0008] This invention employs a gene mining strategy to screen for an aldoxime dehydratase gene derived from *Pseudomonas putida* F1 from the GenBank database. The nucleotide sequence is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2. A recombinant expression plasmid, pRSF-OxdF1, was constructed. A mutant library was constructed and screened. This recombinant plasmid can heterologously and efficiently express the soluble target protein in *Escherichia coli*, exhibiting high catalytic activity in the dehydration of 2,5-furandicarboxaldehyde dioxime to synthesize 2,5-dicyanfuran.

[0009] Secondly, the present invention provides a nucleic acid sequence encoding the above-mentioned aldoxime dehydratase mutant.

[0010] Thirdly, the present invention provides an expression vector comprising the above-mentioned nucleic acid sequence.

[0011] Fourthly, the present invention provides a host cell, such as an Escherichia coli cell, containing the above-described expression vector.

[0012] Fifthly, the present invention provides the application of the above-mentioned aldoxime dehydratase mutant in the catalytic synthesis of dinitrile compounds, wherein the catalytic substrate is any one of succinal dioxime, glutaraldehyde dioxime, 2,5-furandicarboxaldehyde dioxime, o-phthalaldehyde dioxime, iso-phthalaldehyde dioxime, and 2,6-pyridinedicarboxaldehyde dioxime.

[0013] Sixthly, the present invention provides a method for catalytic synthesis of dinitrile compounds, comprising the following steps:

[0014] Genetically engineered bacteria were constructed by introducing the gene encoding an aldoxime dehydratase mutant into host cells; the aldoxime dehydratase mutant was described above.

[0015] Using a dialdehyde oxime compound as a substrate, genetically engineered bacteria were added as a catalyst, and the reaction was carried out at 25-40°C for 2.5-5 hours. After the reaction was completed, the dinitrile compound was isolated and purified from the reaction system. The substrate was any one of succinal dioxime, glutaraldehyde dioxime, 2,5-furandicarboxaldehyde dioxime, o-phthalaldehyde dioxime, iso-phthalaldehyde dioxime, and 2,6-pyridinedicarboxaldehyde dioxime.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] 1. This invention successfully obtained a mutant TU1 with higher substrate specificity and catalytic efficiency by constructing and screening site-directed mutants of the OxdF1 enzyme. This mutant not only efficiently catalyzes the formation of the target product 2,5-dicyanfuran, but also exhibits excellent catalytic conversion ability for structurally similar substrates such as succinal dioxime and glutaraldehyde dioxime. Compared to the natural enzyme, the mutant shows significantly enhanced selectivity and conversion rate in multi-substrate systems, achieving broader substrate adaptability.

[0018] 2. This invention is based on bio-enzymatic catalysis. Compared with organic synthesis DCF, this invention has the advantages of not using expensive metal catalysts and requiring a harsh anhydrous and oxygen-free environment, thus reducing economic and time costs.

[0019] 3. The catalytic reaction of this invention is highly efficient, carried out under mild conditions, and only requires an aqueous phase. The pollution caused to the environment is negligible, which is in line with the concept of "green chemistry" and avoids the problems of large amount of metal catalyst and many by-products in traditional oxidation methods. Attached Figure Description

[0020] Figure 1 shows the HPLC chromatogram of the synthesis of 2,5-furandicarboxaldehyde from 5-hydroxymethylfurfural.

[0021] Figure 2 shows the HPLC chromatogram of the synthesis of 2,5-furandicarboxaldehyde dioxime from 2,5-furandicarboxaldehyde.

[0022] Figure 3 is a schematic diagram of the substrate specificity of the double mutant TU1.

[0023] Figure 4 shows the HPLC chromatogram of the synthesis of 2,5-dicyanfuran from 2,5-furan dialdehyde dioxime.

[0024] Figure 5 shows the 1H NMR spectrum of 2,5-dicyanfuran. Detailed Implementation

[0025] To facilitate understanding of the technical solution of this invention, the following detailed experimental examples further illustrate the redesign strategy of the OxdF1 substrate channel and the catalytic characteristics of its aldoxime dehydratase mutant. The following examples provide a more detailed description of the invention. The plasmids, restriction enzymes, ligases, PCR enzymes, column DNA extraction kits, and DNA gel recovery kits used in these examples are all commercially available products, and the specific operations were performed according to the kit instructions. PCR, nucleic acid agarose gel electrophoresis, protein SDS-PAGE gel electrophoresis, heat shock transformation, and preparation of competent cells were all performed. Sequencing of plasmids and DNA products, as well as gene synthesis, were all completed by Beijing Qingke Biotechnology Co., Ltd.

[0026] In this embodiment of the invention, the liquid phase and gas phase analysis methods are as follows:

[0027] Liquid chromatography (LC) methods: High-performance liquid chromatography (HPLC, Waters H-Class, USA) and a C18 reversed-phase column (5 μm, 4.6 mm × 250 mm, Pursuit 5, Agilent) were used for determination, with the column temperature maintained at 30℃. The detection conditions for the preparation of DFF from HMF were determined at a wavelength of 280 nm, with a mobile phase of solvent A (0.029% TFA, v / v) and solvent B (acetonitrile) in a 60:40 ratio, and a flow rate of 0.3 mL / min. The detection conditions for the preparation of DFFD from DFF were determined at a wavelength of 280 nm, with a mobile phase of solvent A and solvent B in a v / v ratio of 80:20, and a flow rate of 0.3 mL / min. The detection conditions for the preparation of DCF from DFFD were determined at a wavelength of 242 nm, with a mobile phase of solvent A and solvent B in a 30:70 v / v ratio, and a flow rate of 0.4 mL / min.

[0028] Gas chromatography method: Gas chromatography (GC, Agilent, USA) and HP-5 capillary column (0.1 μm, 30 m × 0.25 mm) were used. The injection port and detector temperatures were 220 °C and 250 °C, respectively. The program was held at 35 °C for 2 min, and then increased to 200 °C at a rate of 5 °C / min.

[0029] The present invention will be further described below.

[0030] Example 1: Construction of OxdF1 mutant library

[0031] Before constructing the mutant library, to assess the feasibility of basal channel engineering and broaden the substrate spectrum of Oxd, this invention selected OxdF1 from Pseudomonas putida F1 as a model protein. Using a homology modeling strategy, and with OxdRE (PDB: 3A16) from Rhodococcus N-771 as a template, the three-dimensional complex structure of OxdF1 and heme groups was obtained. Subsequently, the substrate DFFD was docked into the catalytic pocket of OxdF1 to determine its preliminary binding mode with the catalytic residues.

[0032] Furthermore, the substrate channel structure of OxdF1 was analyzed using the CAVER online tool. The results showed that the T2 tunnel is most likely the substrate transport channel, with an elliptical entrance (3.6 × 6.5 Å) and a bottleneck region composed of amino acid residues such as M29, L145, A147, F306, and L318. Based on the T2 channel conformation, traction molecular dynamics (SMD) was used to simulate the energy changes of the substrate as it passes through the channel, screening out key amino acids affecting substrate migration efficiency, providing a basis for subsequent construction of mutant libraries.

[0033] The nucleotide sequence of OxdF1 is shown in SEQ ID NO.1:

[0034]

[0035] The amino acid sequence of OxdF1 is shown in SEQ ID NO.2:

[0036] MESAIDKHLVCPRTLSRRVPDDYQPPFPMWVGRADEQLTQVVMAYLGVQYRGDGQRERALQAMREILGSFSLTDGPLTHDLTHHTDSSGYDNLMIVGYWKDAGAYCRWLRSPEVDGWWSSPQRLNDGLGYYREITAPRAEQFETLYAFQNDLPGVGAIMDNTSGEIEEHGYWGSMR DRFPVSQTDWMNPNGELRVVAGDPAKGGRVVVLGHDNIALIRSGQDWATAEAAERSLYLDEILPTLQDGMDFLRDNGQPLGCYSNRFVRNIDADGNLLDMSYNIGHWRSLEKLERWAESHPTHLRIFVTFFRVAAGLEKLRLYHEVSVSDASSQVFEYINCHPHTGMLRDAKVSSN

[0037] Using recombinant plasmid pRSF-OxdF1 as a template, PCR-mediated site-directed mutagenesis was performed with DNA polymerase (Takara, PrimeSTAR Max). The primer sequences used are shown in Table 1. The PCR reaction system (total volume 50 μL) was as follows: 25 μL PrimeSTAR Max DNA polymerase (2× concentration), 19 μL enzyme-free purified water (ddH2O), 2 μL of each primer (final concentration 10 pmol), and 1 μL of template DNA. The PCR reaction program was set as follows: initial denaturation was performed at 98℃ for 2 minutes; followed by 35 cycles, each cycle consisting of three steps: denaturation at 98℃ for 10 seconds, annealing at 56℃ for 30 seconds, and extension at 72℃ for 1 minute; finally, final extension was performed at 72℃ for 15 minutes. The PCR product was digested and the template was removed by restriction endonuclease Dpn I at 37°C. The product was then transformed into E. coli BL21(DE3) competent cells, and single colonies were picked and cultured. Success was confirmed by gene sequencing.

[0038] Table 1 OxdF1 mutant primers

[0039]

[0040] Example 2: Screening of OxdF1 mutant libraries

[0041] The mutant strain was inoculated into 20 mL of Luria-Bertani (LB) medium with 50 μg / mL kanamycin as antibiotic, and cultured at 37°C and 220 rpm. When the bacterial concentration (OD) 600 When the cytotoxicity reaches 0.6–0.8, IPTG (final concentration 0.4 mM) is added, and induction is performed at 18°C ​​for 15 h. After induction, cells are collected by centrifugation at 8000×g, 4°C, washed, and resuspended in 2 mL of potassium phosphate buffer (50 mM, pH 7.0). The reaction mixture consists of 1 mL of 800 μL cell suspension and 200 μL of DFFD stock solution (final concentration 10 mM, 10% DMSO as a co-solvent). This mixture is placed in a constant-temperature metal bath and shaken at 30°C, 600 rpm for 0.5 h. The reaction is terminated by centrifugation at 4°C, 12000×g for 10 min. The supernatant is diluted, filtered through a 0.22 μm filter, and analyzed by high-performance liquid chromatography (HPLC, Waters).

[0042] After screening, a double mutant with high catalytic activity was obtained and named TU-1. This mutant has a double mutation at the M29A / L318F site based on SEQ ID NO.2.

[0043] Example 3: Enzymatic properties of OxdF1 and its mutants

[0044] This embodiment presents a detailed study on the enzymatic properties and substrate specificity of the screened double mutant TU-1 (and wild-type OxdF1 as a control).

[0045] The activity of OxdF1 was characterized by detecting the amount of DFFD converted to DCF. The reaction mixture (0.5 mL) contained different concentrations of DFFD (1–12 mM), PPB buffer (50 mM, pH 7.0), and an appropriate amount of enzyme, with 10% (v / v) ethanol as a co-solvent. The reaction was carried out at 30 °C and 600 rpm for 5 min, and the reaction was terminated by adding 0.5 mL of acetonitrile. After centrifugation at 12000 × g for 10 min, the mixture was filtered through a 0.22 μm organic filter. The concentration of the reaction product was detected by high-performance liquid chromatography (HPLC, Waters). One unit (U) of Oxd activity was defined as the amount of enzyme that catalyzes the formation of 1 μmol of 2,5-dicyanfuran per minute in PPB. Specific activity was defined as enzyme units per milligram of protein (U / mg protein). The kinetic parameters of the OxdF1 mutant were determined by measuring the enzyme activity of the OxdF1 mutant at different concentrations of DFFD.

[0046] The results are shown in Table 2.

[0047] Table 2 Kinetic parameters of the OxdF1 mutant using DFFD as a substrate.

[0048]

[0049] Example 4: Selective oxidation of HMF

[0050] This embodiment studies the selective oxidation of HMF to synthesize 2,5-furandicarboxaldehyde (DFF) in different organic solvents using Cu(NO3)2 and TEMPO as catalysts under normal pressure. The HPLC chromatogram of the conversion of 5-hydroxymethylfurfural to 2,5-furandicarboxaldehyde is shown in Figure 1.

[0051] HMF (500 mM, 126 mg), Cu(NO3)2·3H2O (37.5 mM, 18.1 mg), and TEMPO (25 mM, 7.8 mg) were added to a 10 mL reaction system and dissolved in acetic acid, acetonitrile, and ethyl acetate (2 mL each), respectively. The cap was then punctured to allow air circulation. The reaction mixture was magnetically stirred at 50 °C, and the consumption of reactants and the formation of substrates were monitored using thin-layer chromatography (TLC). After the reaction was complete, impurities were washed away with a saturated solution of ethylenediaminetetraacetic acid (EDTA).

[0052] The results are shown in Table 3:

[0053] Table 3. Effects of solvent and temperature on HMF conversion and DFF yield a

[0054]

[0055] Note: a. Reaction conditions: HMF (500 mM, 126 mg), Cu(NO3)2·3H2O (37.5 mM, 18.1 mg), and TEMPO (25 mM, 7.8 mg); reaction solvent (2 mL). b. Conversion and yield were determined by HPLC. c. Selectivity (DFF) = Yield (DFF) / Conversion (HMF).

[0056] The results showed that almost all HFM (0.2 mM) was oxidized to DFF in acetonitrile (MeCN), ethyl acetate (EtOAc), and dichloromethane (DCM), with a selectivity of 99%. Subsequently, after separation of the organic phase, it was mixed with an aqueous phase containing hydroxylamine hydrochloride and sodium carbonate for subsequent preparation of DFFD.

[0057] Example 5: Preparation of DFFD by oxime oxidation of DFF

[0058] 2,5-Furfural dioxime (DFFD) was prepared by condensation of 2,5-furandicarboxaldehyde (DFF) with hydroxylamine hydrochloride (NH2OH·HCl), and its HPLC chromatogram is shown in Figure 2. The above reaction solution was diluted to 5 mL, and 5 mL of Na2CO3 (3.3 eq, 660 mM) and NH2OH·HCl (2.2 eq, 440 mM) aqueous solution was added to form a two-phase system. The reaction mixture was reacted at 30 °C for 3 h, and the reaction progress was monitored by TLC. After the reaction was completed, the two phases were separated, extracted three times with ethyl acetate, dried over anhydrous magnesium sulfate, filtered, and rotary evaporated to obtain the target product DFFD. The 2,5-Furfural dioxime was then determined based on 1H NMR spectroscopy (MNR-1H NMR ... 1 The contents of the three isomers (Z,Z-, Z,E- and E,E-) of the product DFFD were determined to be 70.0:27.7:2.3.

[0059] Example 6: Enzymatic Dehydration Synthesis of DCF

[0060] This study investigated the catalytic ability of the double mutant TU1. TU1 preferentially catalyzes both the Z,Z- and Z,E-DFFD configurations, achieving complete conversion to DCF within 3 hours with a yield of approximately 98%. However, this mutant still cannot catalyze the E,E- isomer.

[0061] The enzymatic catalysis was carried out in an aqueous environment. Dimethyl sulfoxide (DMSO, 10%, v / v) was used as a co-solvent to overcome the insolubility of the substrate DFFD (final concentration 50 mM) and was mixed with a whole-cell suspension of recombinant *E. coli* OxdF1 (25 mg / mL, wet weight) in 3 mL of PPB buffer (50 mM, pH 7.0). After stirring the reaction mixture at room temperature for 1 h, an equal volume of bacterial suspension was added, and the reaction was continued for another 1.5 h. The reaction was then terminated by centrifugation at 12000 × g for 5 min to remove the *E. coli* cells. The concentration of DCF in the supernatant was determined by high-performance liquid chromatography (HPLC).

[0062] The results showed that the conversion rate of DFFD was 98%, and the selectivity of DCF was greater than 99%. This result demonstrates the potential of using engineered Oxd to prepare DCF and related aromatic dionitriles.

[0063] Meanwhile, the substrate specificity of mutant TU-1 for other dialdehyde oximes was investigated. The solubilizer DMSO (10%, v / v) overcame the insolubility of dialdehyde oxime substrates (final concentration 20 mM) such as succinal dioxime, and reacted with recombinant *E. coli* OxdF1 whole-cell suspension (25 mg / mL, cell wet weight) in 3 mL PPB buffer (50 mM, pH 7.0). The results showed that mutant TU-1 has a broad substrate range. It not only retained its natural catalytic ability for aliphatic dioximes but also efficiently catalyzed the conversion of aromatic dioximes to their corresponding dionitriles, with aliphatic substrate conversion rates greater than 98%, including succinal dioxime and glutaraldehyde dioxime. For 2,5-furandicarboxaldehyde dioxime and isophthalaldehyde dioxime, the 20 mM substrate was dehydrated by the mutant TU-1 to generate 2,5-dicyanfuran and 1,3-dicyanophenylene, with a conversion rate exceeding 95%.

[0064] The specificity of mutant TU-1 for other aldehyde oxime substrates is shown in Figure 3. The HPLC chromatogram of the conversion of 2,5-furandialdehyde dioxime to 2,5-dicyanfuran is shown in Figure 4. 1 The H NMR spectrum is shown in Figure 5.

[0065] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. An aldoxime dehydratase mutant, characterized in that, They mutated methionine M at position 29 of the wild-type aldoxime dehydratase of SEQ ID NO:2 to alanine A and leucine L at position 318 to phenylalanine F, and designated it as TU-1.

2. A nucleic acid sequence, characterized in that, Encodes the aldoxime dehydratase mutant as described in claim 1.

3. An expression carrier, characterized in that, It contains the nucleic acid sequence as described in claim 2.

4. A host cell, characterized in that, It includes the expression vector as described in claim 3.

5. The application of the aldoxime dehydratase mutant as described in claim 1 in the catalytic synthesis of dinitrile compounds, characterized in that, The catalytic substrate is any one of succinal dioxime, glutaraldehyde dioxime, 2,5-furandicarboxaldehyde dioxime, o-phthalaldehyde dioxime, iso-phthalaldehyde dioxime, and 2,6-pyridinedicarboxaldehyde dioxime.

6. A method for catalytic synthesis of dinitrile compounds, characterized in that, Includes the following steps: The gene encoding the aldoxime dehydratase mutant as described in claim 1 was introduced into a host cell to construct a genetically engineered bacterium; using a dialdehyde oxime compound as a substrate, the genetically engineered bacterium was added as a catalyst, and the reaction was carried out at 25-40°C for 2.5-5 hours; after the reaction was completed, the dinitrile compound was isolated and purified from the reaction system.

7. The method according to claim 6, characterized in that, The substrate is any one of succinal dioxime, glutaraldehyde dioxime, 2,5-furandicarboxaldehyde dioxime, o-phthalaldehyde dioxime, iso-phthalaldehyde dioxime, and 2,6-pyridinedicarboxaldehyde dioxime.

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