A method for the catalytic production of aromatic acids from aromatic aldehydes using oxidized artificial nicotinamide co-factors

Abstract

The application discloses a method for biocatalyzing aromatic aldehyde into aromatic acid by using oxidized artificial nicotinamide cofactor. The method uses aromatic aldehyde compounds as substrates, uses aldehyde dehydrogenase and oxidized artificial nicotinamide cofactor as a catalytic system, and generates aromatic acid compounds through catalytic reaction in a solvent. The method is convenient to operate, has high product yield, and has a good industrial application prospect in the field of biocatalytic preparation of aromatic acid. In the method for biocatalyzing aromatic aldehyde into aromatic acid by using oxidized artificial nicotinamide cofactor, the cofactor used is artificially synthesized and is not a natural nicotinamide cofactor which is expensive, so that the production cost of aromatic acid from aromatic aldehyde is reduced.

Description

Technical Field

[0001] This invention relates to the field of biosynthesis technology, specifically to a method for catalyzing the formation of corresponding aromatic acids from aromatic aldehydes using oxidized artificial nicotinamide as a cofactor. Background Technology

[0002] Aromatic acids (such as benzoic acid, salicylic acid, cinnamic acid, and their derivatives) are important structural units in pharmaceuticals, fragrances, pesticides, and polymer materials. Traditional chemical synthesis methods often rely on harsh conditions (high temperature and pressure, strong acids and bases) and precious metal catalysts, resulting in environmental burdens. Biocatalysis technology offers a promising alternative route for the green synthesis of these compounds. Its core lies in utilizing the specific catalytic capabilities of microorganisms or enzymes to achieve highly selective functional group transformation of aromatic rings under mild conditions. Biocatalytic synthesis of aromatic acids mainly relies on two strategies: first, whole-cell transformation by microorganisms, using natural or engineered microorganisms (such as Pseudomonas, Rhodococcus, and yeast) to progressively oxidize inexpensive aromatic substrates (such as toluene, styrene, and lignin derivatives) into the target acid through their intrinsic metabolic pathways; second, enzyme catalysis, using isolated or immobilized specific enzymes (such as monooxygenases, dehydrogenases, and peroxidases) to carry out key reaction steps. Among these, enzymes involved in oxidation reactions often require NAD(P). + Cofactors and cost-effective cofactor regeneration systems (such as enzyme coupling, photocatalysis, and electrochemical regeneration) are involved. Currently, in the biocatalytic methods for aromatic acid compounds, the oxidized natural nicotinamide cofactor NAD(P) is used. + The high price of raw materials leads to high costs, and there are few reports on the use of artificially synthesized oxidized nicotinamide cofactors in oxidase reactions. Therefore, finding new green methods for the biocatalytic synthesis of aromatic acid compounds is of significant scientific and social value for promoting the green upgrading of existing chemical processes and strengthening environmental protection.

[0003] Aromatic aldehydes are important chemical raw materials, characterized by their ease of acquisition and low price. They are used as raw materials for the synthesis of aromatic acids, resulting in significant economic benefits. Summary of the Invention

[0004] Purpose of the invention: To address the shortcomings of existing technologies, this invention provides a method for catalyzing the formation of aromatic acids from aromatic aldehydes using oxidized artificial nicotinamide as a cofactor.

[0005] To address the aforementioned technical problems, this invention discloses a method for catalyzing the formation of aromatic acids from aromatic aldehydes using an oxidized synthetic nicotinamide cofactor. The method utilizes aromatic aldehyde compounds as substrates and an artificially synthesized oxidized synthetic nicotinamide cofactor to catalyze the reaction via aldehyde dehydrogenase to generate aromatic acid compounds. The specific technical solution is as follows: This invention provides a method for catalyzing the formation of aromatic acids from aromatic aldehydes using oxidized artificial nicotinamide as a cofactor. The method uses aromatic aldehyde compounds as substrates and aldehyde dehydrogenase and oxidized artificial nicotinamide as a catalytic system to generate aromatic acid compounds via a catalytic reaction in a solvent. In some embodiments of this invention, the reaction is carried out in a one-pot manner by adding each component to the catalytic system.

[0006] The aromatic aldehyde compounds include any one of benzaldehyde, phenylacetaldehyde, phenylpropionaldehyde, or cinnamaldehyde; the aromatic acid compounds include any one of benzoic acid, phenylacetic acid, phenylpropionic acid, or cinnamic acid.

[0007] The oxidized artificial nicotinamide cofactor is selected from any one of b4, b5, or b7. .

[0008] The aldehyde dehydrogenase is selected from any one or a combination of several of the following a1 to a7: a1, derived from the genus *Sphingolipidae* Sphingobium The amino acid sequence of sp. SpALDH1 is G2IMY8 (UniProtKB). a2, derived from Escherichia coli Escherichia coli The amino acid sequence of puuc is UniProtKB, which is P23883. a3, derived from *Trichomonas testis* Comamonas testosteroni The amino acid sequence of Ctvdh1, UniProtKB, is A0A7D3QIR7; a4, derived from E. coli Escherichia coli The amino acid sequence of UniProtKB is P37685; a5, derived from Escherichia coli Escherichia coli The amino acid sequence of gabD, UniProtKB, is P25526; a6, derived from Escherichia coli Escherichia coli The amino acid sequence of feaB, UniProtKB, is P80668; a7, derived from *Pseudomonas putida* Pseudomonas putida The amino acid sequence of PpALDH, UniProtKB, is AOA179ROL7.

[0009] The oxidized artificial nicotinamide cofactor is b4, and the aldehyde dehydrogenase is a5.

[0010] The solvent includes a phosphate buffer. In some embodiments of the present invention, the phosphate buffer is a potassium phosphate (Kpi) buffer with a concentration of 50-100 mM and a pH of 6-8. In other embodiments of the present invention, the solvent is a 50 mM Kpi buffer with a pH of 7-8.

[0011] The aldehyde dehydrogenase is catalyzed in the form of recombinant cells, crude enzyme solution, or pure enzyme.

[0012] The aromatic aldehyde compound has an initial concentration of 1-10 mM. In some embodiments of the present invention, the initial concentration of the aromatic aldehyde compound is 5 mM.

[0013] The aldehyde dehydrogenase has an initial concentration of 1-5 U / mL. In some embodiments of the present invention, the initial concentration of the aldehyde dehydrogenase is 3 U / mL. The enzyme activity unit U is the catalytic oxidation of 1 μmol mNAD per minute. + The amount of enzyme required for s.

[0014] The aldehyde dehydrogenase described in this invention can be purchased from the market or prepared using conventional methods. Specifically, the preparation method is as follows: When the aldehyde dehydrogenase exists in the form of recombinant cells, its preparation method is as follows: A nucleic acid molecule capable of expressing aldehyde dehydrogenase is introduced into a host cell, and after induction of expression, recombinant cells are obtained. The host cell is a prokaryotic cell or a eukaryotic cell; the prokaryotic cell is preferably a bacterium, the eukaryotic cell is preferably a yeast cell, and the bacterium is preferably *Escherichia coli*, and more preferably... E. coli BL21(DE3). The nucleic acid molecule capable of expressing aldehyde dehydrogenase is introduced into the host cell in the form of a recombinant vector; the recombinant expression vector is transferred to a suitable microbial host by heat shock transformation, and the recombinant vector is a bacterial plasmid, bacteriophage, yeast plasmid, or retroviral packaging plasmid carrying the aldehyde dehydrogenase encoding gene. The induction expression is performed by adding the inducer IPTG (isopropyl-β-D-thiogalactoside) to the culture system of the genetically engineered strain to a final concentration of 0.1~0.6 mM, and inducing culture at 20~35 ℃ for 12~24 h, preferably at 25 ℃ for 18~20 h.

[0015] When the aldehyde dehydrogenase exists in the form of crude enzyme solution, its preparation method is as follows: lyse the recombinant cells to obtain the crude enzyme solution.

[0016] When the aldehyde dehydrogenase exists in pure enzyme form, its preparation method is as follows: after centrifuging the crude enzyme solution, the supernatant is collected and purified by nickel column chromatography to obtain the pure enzyme.

[0017] The oxidized artificial nicotinamide cofactor has an initial concentration of 1-20 mM. In some embodiments of the present invention, the initial concentration of the oxidized artificial nicotinamide cofactor is 20 mM.

[0018] The catalytic reaction is carried out at a temperature of 25–37 °C for a time of 2–48 h. In some embodiments of the present invention, the reaction temperature is 30 °C and the reaction time is 12 h.

[0019] Beneficial effects: Compared with the prior art, the present invention has the following advantages: 1. This invention provides a novel green biosynthetic route for aromatic acids, using aromatic aldehydes as raw materials to synthesize aromatic acids via aldehyde dehydrogenase ALDH and oxidized artificial nicotinamide as a cofactor. This invention is easy to operate, yields high product rates, and has promising industrial application prospects in the biocatalytic preparation of aromatic acids. 2. In the method of generating corresponding aromatic acids from aromatic aldehydes using artificially synthesized oxidized nicotinamide cofactor, the cofactor used in this invention is artificially synthesized, the synthesis steps are simple, the raw materials are inexpensive, and the production cost of aromatic acid compounds is reduced. Attached Figure Description

[0020] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, and the advantages of the present invention in the above and / or other aspects will become clearer.

[0021] Figure 1 This is the synthetic reaction formula for the cofactor of oxidized artificial nicotinamide. Wherein, X is any one of H or CH3; Y is CH2Ph, CH2Ph-OCH3, n-C4H9, or n-C8H. 17 Any one of CH2Ph-COOH or CH2Ph-COOC2H5.

[0022] Figure 2 This is a schematic diagram of the reaction process of generating corresponding aromatic acids from aromatic aldehydes using artificially synthesized oxidized nicotinamide cofactor according to the present invention. Wherein, R is selected from any one of CH2, C2H4, C2H2, or C3H6. Detailed Implementation

[0023] The present invention will be further described below with reference to the following embodiments. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the present invention.

[0024] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0025] The substrates benzaldehyde, phenylacetaldehyde, phenylpropionaldehyde, and cinnamaldehyde used in the following examples were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; the cofactors used were artificially synthesized, and the raw materials were from Beijing Bailingwei Technology Co., Ltd. and Shanghai Aladdin Biochemical Technology Co., Ltd.

[0026] In the following examples, the reaction solution obtained from the catalytic reaction was processed and quantitatively analyzed by HPLC. Specifically, 200 μL of the reaction solution and 800 μL of methanol were thoroughly mixed, heated in a 95°C water bath for 5 min, centrifuged at 8000 rpm for 1 min, the supernatant was collected and filtered through a 0.22 μm filter membrane, and the filtrate was analyzed by HPLC. The HPLC detection method was as follows: high performance liquid chromatography was performed on an Agilent system using an autosampler. The chromatographic column was a C18 column (5 μm, 4.6 × 250 mm). A diode array detector (DAD) was used to detect the analyte at a wavelength of 261 nm. The mobile phase was pure water and acetonitrile at a volume ratio of 3:1, with isogradient elution at a flow rate of 0.4 mL / min. The test temperature was 25°C, and the detection wavelength was 254 nm.

[0027] In the quantitative experiments in the following examples, three replicate experiments were set up, and the average value of the results was taken.

[0028] The product yield is calculated as: actual product yield / theoretical product yield × 100%.

[0029] Example 1 Expression of aldehyde dehydrogenase The aldehyde dehydrogenases (ALDH) used in this invention are shown in Table 1. After being synthesized by a company, the various aldehyde dehydrogenases shown in Table 1 were introduced into *E. coli*. E. coli The target strain was obtained from BL21(DE3). Aldehyde dehydrogenase was expressed and purified using the following methods: 1. Activation and Expansion Culture of the Strains: The target strain was removed from its -80°C cryopreservation and thawed at 4°C. 150 µL of the bacterial culture was transferred to an Erlenmeyer flask containing 20 mL of sterile LB broth, and kanamycin was added to a final concentration of 50 µg / mL. The culture was incubated at 37°C with shaking at 200 rpm for 12 hours to complete the primary culture. The entire primary culture was then transferred to a fermentation flask containing 380 mL of sterile LB broth, and kanamycin (50 µg / mL) was added. The culture was continued at 37°C with shaking at 200 rpm until the bacterial culture reached its OD value. 600The concentration was increased to 0.6-0.8. Isopropyl-β-D-thiogalactoside (IPTG) was then added to a final concentration of 0.6 mM, and the culture conditions were adjusted to 25 °C and 200 rpm to induce expression of the target protein for 18-20 hours.

[0030] 2. Cell Collection and Washing: After induction, the bacterial suspension was centrifuged at 8500 rpm for 6 min at 4 °C, and the cell pellet was collected. The bacterial sludge was then resuspended and washed three times with 50 mM KPi buffer (pH 7.0). Finally, the washed cells were stored at 4 °C for later use.

[0031] 3. Preparation of purified enzyme: Take the bacterial precipitate obtained from washing in step 2 and resuspend it in 20 mL of 50 mM potassium phosphate buffer (KPi, pH 7.0). Place the resuspended solution in an ice-water bath and use an ultrasonic cell disruptor for disruption. Set the parameters as follows: working time 3 seconds, interval time 5 seconds, power 300 W, and total disruption time 20 minutes. After disruption, centrifuge the sample at 4 °C and 10,000 rpm for 60 minutes, collect the supernatant, and filter it through a 0.22 μm aqueous filter membrane. The resulting filtrate is the crude enzyme extract. Pack 3 mL of Ni-NTA agarose purification medium into a chromatography column and equilibrate it three times with the above-mentioned KPi buffer. Then, slowly load an appropriate amount of crude enzyme solution and incubate it with gentle shaking at room temperature for 10 minutes to allow the target protein to fully bind with the Ni-NTA agarose purification medium. Use imidazole solutions with concentrations ranging from 20 to 200 mM for gradient elution, and collect the eluted fractions in 50 mL centrifuge tubes. The collected protein solution was further concentrated and desalted using Millipore ultrafiltration centrifuge tubes to obtain a high-purity enzyme solution. The purified enzyme solution was aliquoted into centrifuge tubes and stored at -80°C for later use.

[0032] Table 1. Sources of various aldehyde dehydrogenases

[0033] Example 2 Synthesis of Oxidized Artificial Nicotinamide Cofactor The oxidized artificial nicotinamide cofactors used in this invention include b1~b30, specifically as follows:

[0034]

[0035] Oxidized artificial nicotinamide cofactor (mNAD) + The synthesis of s) uses nicotinamide or its analogues as raw materials, and the synthetic formula is as follows: Figure 1As shown. The specific synthesis method is as follows: Nicotinamide (10 mM) or its analogue is mixed with a haloalkane (Y-Br, 12 mM) in acetonitrile (100 mL), and reacted at 80 °C for 12 h to obtain oxidized mNAD. + s, as a cofactor in the subsequent catalytic system. Oxidized mNAD + mNADHs were obtained by reacting s under alkaline conditions (NaHCO3) with a strong reducing agent (sodium hyposulfite Na2S2O4) at room temperature for 3-4 h.

[0036] Example 3: Aldehyde dehydrogenase combined with oxidized artificial nicotinamide cofactor catalyzes the production of benzoic acid from benzaldehyde. The aldehyde dehydrogenase used in this embodiment is the pure enzyme shown in Example 1, and the oxidized synthetic nicotinamide cofactor used is shown in Example 2. This embodiment uses an aldehyde dehydrogenase combined with an oxidized synthetic nicotinamide cofactor to catalyze the production of benzoic acid from benzaldehyde. The catalytic reaction is specifically shown below: A complete biocatalytic reaction system was constructed by sequentially adding 50 mM Kpi buffer (pH 7.0), 5 mM benzaldehyde, 20 mM oxidized artificial nicotinamide cofactor (including any one of b1-b8 described in Example 2), and 3 U / mL purified aldehyde dehydrogenase enzyme (including any one of puuc, Ctvdh1, aldB, gabD, or SpALDH) to a reaction vessel. The reaction system was reacted at 30 °C for 12 h to obtain the reaction solution. The reaction solution was then processed and analyzed by HPLC. The names of the oxidized artificial nicotinamide cofactors corresponding to b1-b8 described in Example 2 are shown in Table 2. The enzyme activity unit U of the aldehyde dehydrogenase described in this invention is the catalytic oxidation of 1 μmol mNAD per minute. + The amount of enzyme required for s.

[0037] Table 2. Names of Oxidized Artificial Nicotinamide Cofactors Corresponding to Different Serial Numbers

[0038] Some reaction results are shown in Tables 3-7. As can be seen from Tables 3-7, 5-Me-BuNA with serial number b4... + BuNA with serial number b5 + And BNA with serial number b7 + -COOH artificial cofactor combinations with different aldehyde dehydrogenases can catalyze the production of benzoic acid in relatively high yields. Among them, the most effective is 5-Me-BuNA of b4. + The combined aldehyde dehydrogenase gabD can catalyze the highest yield of benzoic acid, with a yield of 91.38%, as shown in Table 6.

[0039] Table 3 Results of benzoic acid production catalyzed by different artificial cofactors in combination with aldehyde dehydrogenase puuc

[0040] Table 4 Results of benzoic acid production catalyzed by different artificial cofactors in combination with aldehyde dehydrogenase Ctvdh1

[0041] Table 5 Results of benzoic acid production catalyzed by different artificial cofactors in combination with aldehyde dehydrogenase aldB.

[0042] Table 6 Results of benzoic acid production catalyzed by different artificial cofactors in combination with aldehyde dehydrogenase gabD

[0043] Table 7 Results of benzoic acid production catalyzed by different artificial cofactors in combination with the aldehyde dehydrogenase SpALDH

[0044] Example 4: Catalytic formation of aromatic acid compounds from aromatic aldehydes This embodiment uses aldehyde dehydrogenase gabD and oxidized artificial nicotinamide cofactor b4 (5-Me-BuNA). + Catalyzing the formation of aromatic acids from aromatic aldehydes, the general reaction formula is as follows: Figure 2 As shown.

[0045] 1. Catalytic conversion of phenylpropionaldehyde to phenylpropionic acid: The specific steps are as follows: Add 50 mM MKpi buffer (pH 7.0), 5 mM phenylpropionaldehyde, and 20 mM 5-Me-BuNA to the reaction vessel sequentially. + A complete biocatalytic reaction system was formed by combining gabD, an aldehyde dehydrogenase, with an initial concentration of 3 U / mL. The reaction system was incubated at 30 °C for 12 h to obtain the reaction solution. The reaction solution was then processed and analyzed by HPLC. The results showed that the yield of phenylpropionaldehyde reached 8.92%, indicating that gabD and the oxidized artificial nicotinamide cofactor 5-Me-BuNA were effective. + It can catalyze the formation of phenylpropionic acid from phenylpropionaldehyde.

[0046] 2. Catalytic conversion of phenylacetaldehyde to phenylacetic acid: The specific steps are as follows: Add 50 mM Mkpi buffer (pH 8.0), 5 mM phenylacetaldehyde, and 20 mM 5-Me-BuNA to the reaction vessel sequentially. +A complete biocatalytic reaction system was constructed using aldehyde dehydrogenase gabD at an initial concentration of 3 U / mL. The reaction system was reacted at 30 °C for 12 h to obtain the reaction solution. The reaction solution was then processed and analyzed by HPLC. The results showed that the yield of phenylacetaldehyde reached 38.08%, indicating that aldehyde dehydrogenase gabD and the oxidized artificial nicotinamide cofactor 5-Me-BuNA were effective. + It can catalyze the formation of phenylacetaldehyde into phenylacetic acid.

[0047] 3. Catalytic conversion of cinnamaldehyde to cinnamic acid: The specific steps are as follows: Add 50 mM KPI buffer (pH 7.0), 5 mM cinnamaldehyde, and 20 mM 5-Me-BuNA to the reaction vessel sequentially. + A complete biocatalytic reaction system was formed by combining aldehyde dehydrogenase gabD (initial concentration 3 U / mL) with purified gabD. The reaction system was reacted at 30℃ for 12 h to obtain the reaction solution. The reaction solution was then processed and analyzed by HPLC. The results showed that the yield of phenylpropionaldehyde reached 7.92%, indicating that aldehyde dehydrogenase gabD and the oxidized artificial nicotinamide cofactor 5-Me-BuNA were effective. + It can catalyze the formation of cinnamaldehyde into cinnamic acid.

[0048] This invention provides a method for catalyzing the formation of aromatic acids from aromatic aldehydes using oxidized synthetic nicotinamide as a cofactor. Many methods and approaches exist for implementing this technical solution; the above description is merely a preferred embodiment of the invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of this invention, and these improvements and modifications should also be considered within the scope of protection of this invention. All components not explicitly stated in this embodiment can be implemented using existing technologies.

Claims

1. A method for catalyzing the formation of aromatic acids from aromatic aldehydes using oxidized synthetic nicotinamide as a cofactor, characterized in that, Using aromatic aldehydes as substrates and aldehyde dehydrogenase and oxidized artificial nicotinamide as cofactors as catalytic systems, aromatic acid compounds are generated through catalytic reactions in solvents. The aromatic aldehyde compounds include any one of benzaldehyde, phenylacetaldehyde, phenylpropionaldehyde, or cinnamaldehyde; the aromatic acid compounds include any one of benzoic acid, phenylacetic acid, phenylpropionic acid, or cinnamic acid. The oxidized artificial nicotinamide cofactor is selected from any one of b4, b5, or b7: 。 2. The method according to claim 1, characterized in that, The aldehyde dehydrogenase is selected from any one or a combination of several of the following a1 to a7: a1, derived from the genus *Sphingolipidae* Sphingobium The amino acid sequence of sp. SpALDH1 is G2IMY8 (UniProtKB). a2, derived from Escherichia coli Escherichia coli The amino acid sequence of puuc is UniProtKB, which is P23883. a3, derived from *Trichomonas testis* Comamonas testosteroni The amino acid sequence of Ctvdh1, UniProtKB, is A0A7D3QIR7; a4, derived from E. coli Escherichia coli The amino acid sequence of UniProtKB is P37685; a5, derived from Escherichia coli Escherichia coli The amino acid sequence of gabD, UniProtKB, is P25526; a6, derived from Escherichia coli Escherichia coli The amino acid sequence of feaB, UniProtKB, is P80668; a7, derived from *Pseudomonas putida* Pseudomonas putida The amino acid sequence of PpALDH, UniProtKB, is AOA179ROL7.

3. The method according to claim 2, characterized in that, The oxidized artificial nicotinamide cofactor is b4, and the aldehyde dehydrogenase is a5.

4. The method according to claim 1, characterized in that, The solvent mentioned includes phosphate buffer.

5. The method according to claim 4, characterized in that, The phosphate buffer is a potassium phosphate buffer with a concentration of 50-100 mM and a pH of 6-8.

6. The method according to claim 1, characterized in that, The aldehyde dehydrogenase is catalyzed in the form of recombinant cells, crude enzyme solution, or pure enzyme.

7. The method according to claim 1, characterized in that, The aromatic aldehyde compound has an initial concentration of 1~10 mM.

8. The method according to claim 1, characterized in that, The aldehyde dehydrogenase has an initial concentration of 1-5 U / mL.

9. The method according to claim 1, characterized in that, The oxidized artificial nicotinamide cofactor has an initial concentration of 1~20 mM.

10. The method according to claim 1, characterized in that, The catalytic reaction is carried out at a temperature of 25-37 °C for 2-48 h.

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