Electrochemical-biological oxidoreductase cascade catalysis method

Abstract

The invention belongs to the technical field of bioengineering and electrochemical chemical engineering, and particularly relates to an electrochemical-biological oxidoreductase cascade catalysis method. The electrochemical-biological combined synthesis technology is adopted, threonine deaminase is accelerated and promoted to catalyze threonine in an electrolysis mode, hydrogen peroxide generated in the oxidation process is promoted to be decomposed to generate oxygen and water, and therefore the effect of catalase is completely replaced. Meanwhile, in the electrolytic tank, part of water molecules in the reaction system can generate oxygen at the anode of the electrolytic tank, and oxygen is provided for DAAO; and when a working electrode is adjusted from a platinum electrode to foam titanium and anode and cathode conversion is carried out, NAD < + > can be effectively reduced to NADH, and a coenzyme regeneration system is provided for conversion of 2-ketobutyric acid to L-2-aminobutyric acid and reduction of PPO to L-PPT. A technical route of adding catalase, alcohol dehydrogenase and isopropanol required by the L-PPT obtained by splitting the D, L-PPT is abandoned.

Description

Technical Field

[0001] This invention belongs to the fields of bioengineering and electrochemical chemical engineering, specifically relating to an electrochemical-biological redox enzyme cascade catalytic method. Background Technology

[0002] Electrochemical technology has shown great potential as a clean, precise, and controllable electron source, aiming to fundamentally revolutionize electron transport pathways by directly injecting electrons into catalytic systems through electrodes. However, achieving efficient and specific direct electron transport faces significant challenges, the core of which lies in optimizing the electron transport efficiency and selectivity at the electrode-biointerface and avoiding disturbances to the enzyme's native conformation by external electric fields.

[0003] Against this backdrop, electroenzyme combinatorial catalysis has emerged, combining the programmability of electrochemical reactions with the stereospecificity of enzyme catalysis, exhibiting unique advantages. On the one hand, electroenzyme combinatorial catalysis achieves direct and directional injection of exogenous electrons by precisely matching the energy levels of electrode materials with the redox potentials of enzyme active sites, thereby bypassing the diffusion limitations imposed by traditional chemical mediators, significantly reducing the risk of side reactions, and greatly improving electron transfer efficiency. On the other hand, it spatially decouples the oxidation and reduction steps. Based on the catalytic characteristics of redox enzymes, appropriate electrodes are selected in a two-electrode system to perform oxidation and reduction reactions separately. The electrochemical workstation facilitates electron transfer between the electrodes, enzymes, and substrates, and performs a regeneration cycle of the cofactor reduced nicotinamide adenine dinucleotide (NADH) in the cathode region. The highly selective enzyme steps complete complex chiral transformations, preserving the natural stereospecificity and effectively suppressing the accumulation of non-target products. Furthermore, real-time adjustment of potential / current pulses can dynamically balance enzyme activity and electron transfer under mild conditions, avoiding irreversible damage to enzyme structure caused by high temperature, high pressure or strong oxidizing / reducing agents. This aligns with the concept of green chemistry and provides an innovative paradigm with both theoretical depth and application prospects for cofactor regeneration and synthesis of complex chiral compounds with high atom economy and high stereoselectivity.

[0004] This invention focuses on the synergistic optimization of electro-enzyme combined catalytic processes. It investigates various biocatalytic reactions involving redox enzymes using multiple electrode materials (platinum, titanium, nickel-molybdenum alloy, copper, graphite, etc.) to achieve highly efficient electro-enzyme combined catalytic systems. The biocatalytic systems of this invention select the synthesis of L-2-aminobutyric acid from threonine via synergistic catalysis by threonine deaminase (TDH) and leucine dehydrogenase (LeuDH) and the synthesis of L-glufosinate from D-glufosinate via D-amino acid oxidase (DAAO) and glufosinate dehydrogenase (PpDH) as research subjects, but are not limited to these two biocatalytic reactions. Summary of the Invention

[0005] This invention aims to provide an electrochemical-biological redox enzyme cascade catalytic method, in which threonine is selected to synthesize L-2-aminobutyric acid through the synergistic catalysis of threonine deaminase and leucine dehydrogenase; and D-glufosinate is selected to synthesize L-glufosinate through D-amino acid oxidase and glufosinate dehydrogenase. This method helps to save costs, reduce the generation of by-products, and provides a new strategy for green chemical synthesis.

[0006] To achieve the above-mentioned objectives, the present invention is implemented through the following technical solution: An electrochemical-biological redox enzyme cascade catalytic method includes the following steps: In an electrochemical reaction cell, an electrochemical workstation is used to control the polarity of the working electrode, causing it to alternate between the anode and cathode to promote the catalytic reaction of at least one redox enzyme and achieve the electrochemical regeneration of the coenzyme; wherein the polarity switching of the working electrode is achieved by potential adjustment, thereby synthesizing the target compound.

[0007] This invention achieves precise decoupling and seamless integration of two distinctly different catalytic functions through dynamic switching of electrode polarity. When the working electrode is set as the anode, the resulting hole or oxidative environment efficiently promotes the catalytic oxidation reaction of oxidoreductases (such as D-amino acid oxidase or threonine deaminase). Simultaneously, at a suitable potential, the anode can directly electrolyze the hydrogen peroxide or water generated during the reaction to produce oxygen, thus simultaneously completing oxidation catalysis and in-situ elimination of harmful byproducts in one step, completely eliminating dependence on catalase. Subsequently, via commands from the electrochemical workstation, the polarity of the working electrode is switched to the cathode, at which point the electrode surface transforms into a reducing environment. Through this polarity reversal, the cathode not only provides suitable reduction reaction conditions for subsequent reductases (such as glufosinate dehydrogenase or leucine dehydrogenase), but more importantly, it can also directly and efficiently remove NAD3 from the system. + Electrochemical reduction and regeneration to NADH replaces the complex chemical coenzyme regeneration system required in traditional methods with a programmable electrical signal.

[0008] This invention drives a complete biocatalytic cycle through alternating electrode polarity switching, simplifying the process and significantly reducing raw material costs and operational complexity. Simultaneously, by avoiding the addition of chemicals such as ammonium formate and isopropanol, it reduces the generation of byproducts at the source, making the reaction process cleaner and more environmentally friendly, aligning with the development direction of green chemistry. Furthermore, the precise controllability of electrochemical parameters provides great flexibility for reaction optimization. By optimizing the frequency of polarity switching, the magnitude and waveform of the applied potential, the catalytic characteristics of different enzymes can be dynamically matched, thereby significantly improving the overall efficiency and specificity of the reaction. In addition, this invention integrates the precise control capabilities of electrochemistry with the high efficiency and specificity of enzyme catalysis, successfully unifying the originally discrete and complex multi-reagent system onto a simple and controllable electro-driven platform, contributing to simplified processes, improved efficiency, and the realization of green synthesis.

[0009] Preferably, the oxidoreductase is selected from at least one of dehydrogenase and oxidase.

[0010] Preferably, the dehydrogenase is any one or a combination of two of leucine dehydrogenase and glufosinate dehydrogenase; the oxidase is any one or a combination of two of D-amino acid oxidase and threonine deaminase.

[0011] Preferably, the working electrode is made of any one or more of the following materials: platinum, titanium, nickel-molybdenum alloy, copper, graphite, and gold.

[0012] Preferably, the potential adjustment employs a multi-segment pulsed reaction potential, which includes a combination of at least two of the following: oxidation potential, reduction potential, and hydrogen peroxide decomposition potential.

[0013] Preferably, the cycle period of the pulse voltage of the multi-segment pulsed reactive potential is 0.5~10 min.

[0014] Preferably, the cycle period of the pulse voltage of the multi-segment pulsed reactive potential is 2~5 minutes.

[0015] Preferably, the coenzyme is NAD. + / NADH, the regeneration of the coenzyme is achieved through cathodic reduction.

[0016] Preferably, the reaction medium of the electrochemical reaction cell is a buffer solution with a pH of 7 to 9.

[0017] As a further preferred embodiment, the reaction medium of the electrochemical reaction cell is a Tris-HCl buffer solution with a pH of 7-9.

[0018] Preferably, the catalytic reaction is carried out at a temperature of 35-50°C, a pressure of 1.01 MPa, a rotation speed of 600 rpm / min, and a reaction time of 0.5-8 h.

[0019] Preferably, the target compound is a chiral amino acid or a derivative thereof.

[0020] As a further preferred option, the target compound is L-2-aminobutyric acid or L-glufosinate.

[0021] The application of the electrochemical-biological redox enzyme cascade catalysis method described above in the catalytic synthesis of L-2-aminobutyric acid from threonine.

[0022] This invention utilizes an electrochemical system coupled with threonine deaminase (TDH) and leucine dehydrogenase (LeuDH) to synergistically catalyze the efficient synthesis of L-2-aminobutyric acid from threonine. Using threonine as a substrate, the reaction is first accelerated by threonine deaminase (TDH) at the anode of an electrolytic cell, increasing the efficiency of 2-ketobutyric acid (2-KBA) formation. The NAD+ is then desorbed into the reaction system, and the electrode polarity is reversed by an electrochemical workstation. Since the electrochemical workstation converts the current to a cathode reduction current, it can also efficiently remove an appropriate amount of NAD+. + The NADH is regenerated and then combined with leucine dehydrogenase (LeuDH) to catalyze the synthesis of L-2-aminobutyric acid. In this process, electrochemistry can not only promote the deamination reaction of threonine, but also provide NADH as a coenzyme for leucine dehydrogenase to catalyze 2-ketobutyric acid. This effectively replaces the use of ammonium formate and ammonium formate dehydrogenase in the traditional biological method, which helps to save costs, reduce the generation of by-products, and provides a new strategy for green chemical synthesis.

[0023] The application of the electrochemical-biological redox enzyme cascade catalysis method described above in the catalytic production of L-glufosinate from D-glufosinate.

[0024] This invention uses D-glufosinate (D-PPT) as a substrate. First, it is oxidized by D-amino acid oxidase (DAAO) at the anodic oxidation potential in an electrolytic cell to generate α-keto acid-2-carbonyl-4-(hydroxymethylphosphono)butyric acid (PPO). This not only effectively improves the catalytic efficiency of DAAO but also completely replaces the addition of catalase during the oxidation process. The generated PPO desorbs into the reaction system, and then the electrode polarity is changed by an electrochemical workstation. Since the electrochemical workstation converts the current to a cathode reduction current, it can also efficiently remove an appropriate amount of NAD3. +The NADH is regenerated and then combined with glufosinate dehydrogenase (PpDH) to asymmetricly synthesize L-glufosinate (L-PPT). This process replaces the addition of alcohol dehydrogenase and isopropanol in traditional biocatalytic systems, helping to reduce the formation of byproducts. This invention improves the catalytic efficiency of the oxidation or deammoniation stages of the entire catalytic system by electrochemically coupling redox enzymes to synergistically catalyze biological reactions and provides coenzyme regeneration capacity for the dehydrogenation reduction process. The entire electrochemical-enzymatic synthesis process is simple and provides a new method for the synthesis of L-glufosinate.

[0025] Therefore, the present invention has the following beneficial effects: (1) Traditional routes start from existing threonine and catalytically synthesize L-2-aminobutyric acid through deamination and dehydrogenation reduction; L-glufosinate is obtained from D,L-glufosinate through a resolution method. However, this invention innovatively adopts an electrochemical-biological combined synthesis technology to accelerate the threonine deaminase catalysis of threonine by electrolysis, promoting the decomposition of hydrogen peroxide generated during the oxidation process to produce oxygen and water, thereby completely replacing the role of catalase. At the same time, some water molecules in the reaction system can also generate oxygen at the anode of the electrolytic cell, providing oxygen for the oxidation of D-2-amino-4-[hydroxy(methyl)phosphono]-butyric acid by D-amino acid oxidase; and when the working electrode is changed from a platinum electrode to foamed titanium and the anode and cathode are switched, NAD can be effectively converted. + The process reduces NADH back to provide a coenzyme regeneration system for the conversion of 2-ketobutyric acid (2-KBA) to L-2-aminobutyric acid (L-2-ABA) and α-keto-2-carbonyl-4-(hydroxymethylphosphono)butyric acid (PPO) to L-2-amino-4-[hydroxy(methyl)phosphono]-butyric acid (L-PPT). This method eliminates the need for adding catalase, alcohol dehydrogenase, and isopropanol, which are required in the further separation of D,L-glufosinate (D,L-PPT) to obtain L-glufosinate (L-PPT). (2) The process route of the present invention is green and does not require additional configuration of coenzyme cycle system, effectively avoiding the addition of catalase, ammonium formate, acid dehydrogenase, alcohol dehydrogenase and isopropanol, which helps to reduce the generation of by-products in the synthesis. (3) The process of the present invention is simple and the reaction conditions are mild. Through the coupling of electrochemical and biological enzyme methods, oxidation and reduction can be carried out in the same electrolytic cell. Compared with the existing methods, separation and purification are convenient. Attached Figure Description

[0026] Figure 1 This is a schematic diagram of the electrochemical-biosynthesis of L-glufosinate.

[0027] Figure 2The reaction process diagram for the electrochemical-enzyme combination catalysis of 50mM D-2-amino-4-[hydroxy(methyl)phosphono]-butyric acid (D-PPT) to L-2-amino-4-[hydroxy(methyl)phosphono]-butyric acid (L-PPT) at -0.5V is shown.

[0028] Figure 3 The reaction process diagram for the electrochemical-enzyme combination catalysis of 50mM D-2-amino-4-[hydroxy(methyl)phosphono]-butyric acid (D-PPT) to L-2-amino-4-[hydroxy(methyl)phosphono]-butyric acid (L-PPT) at -0.6V is shown.

[0029] Figure 4 The reaction process diagram for the electrochemical-enzyme combination catalysis of 50mM D-2-amino-4-[hydroxy(methyl)phosphono]-butyric acid (D-PPT) to L-2-amino-4-[hydroxy(methyl)phosphono]-butyric acid (L-PPT) at -0.7V is shown.

[0030] Figure 5 The reaction process diagram for the electrochemical-enzyme combination catalysis of 50mM D-2-amino-4-[hydroxy(methyl)phosphono]-butyric acid (D-PPT) to L-2-amino-4-[hydroxy(methyl)phosphono]-butyric acid (L-PPT) at -0.8V is shown.

[0031] Figure 6 The reaction process diagram for the electrochemical-enzyme combination catalysis of 50mM D-2-amino-4-[hydroxy(methyl)phosphono]-butyric acid (D-PPT) to L-2-amino-4-[hydroxy(methyl)phosphono]-butyric acid (L-PPT) at a voltage of -0.9V.

[0032] Figure 7 The reaction process diagram shows the electrochemical-enzyme combination catalyzing the conversion of 50mM D-2-amino-4-[hydroxy(methyl)phosphono]-butyric acid (D-PPT) to L-2-amino-4-[hydroxy(methyl)phosphono]-butyric acid (L-PPT) after the introduction of pulse potentials with 6 different periods.

[0033] Figure 8 The reaction process diagram shows the electrochemical-enzyme combination catalysis of 50mM D-2-amino-4-[hydroxy(methyl)phosphono]-butyric acid (D-PPT) to L-2-amino-4-[hydroxy(methyl)phosphono]-butyric acid (L-PPT) after the introduction of a multi-segment pulsed potential reaction.

[0034] Figure 9 The efficiency of L-2-aminobutyric acid production using four different metal electrodes at their respective optimal catalytic potentials is shown in the graph. Detailed Implementation

[0035] The present invention will be further described below with reference to the accompanying drawings and specific embodiments. Those skilled in the art will be able to implement the present invention based on these descriptions. Furthermore, the embodiments of the present invention described below are generally only some, not all, of the embodiments of the present invention. Therefore, all other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort should fall within the scope of protection of the present invention.

[0036] Example 1: Obtaining Enzyme Protein Recombinant *E. coli* strains containing genes encoding threonine deaminase, leucine dehydrogenase, D-amino acid oxidase, and glufosinate dehydrogenase, preserved in the laboratory of the College of Bioengineering, Zhejiang University of Technology, were inoculated into LB broth containing 40 μg / mL ampicillin and cultured at 37°C for 8 hours. Then, 2% (v / v) of the inoculum was added to fresh LB broth containing 40 μg / mL ampicillin and cultured at 37°C and 180 rpm for 2 hours. IPTG was then added to the culture medium to a final concentration of 0.2 mM, and the culture was incubated at 18°C ​​for 14 hours. The bacterial cells were then collected by centrifugation at 8000 rpm for 10 minutes. The bacterial cells were ultrasonically disrupted for 90 minutes, centrifuged at 4°C and 8000 rpm for 10 minutes, and the supernatant was used to obtain purified enzymes via nickel column chromatography. The construction process of recombinant *E. coli* engineered bacteria containing a threonine deaminase encoding gene and the nucleotide sequence of the encoding gene are described in patent application CN201510751632.6. The construction process of recombinant *E. coli* engineered bacteria containing a leucine dehydrogenase encoding gene and the nucleotide sequence of the encoding gene are described in patent application CN201610867380.8. The construction process of recombinant *E. coli* engineered bacteria containing a D-amino acid oxidase encoding gene and the amino acid sequence of the encoded protein are described in patent application CN202010191945.1. The construction process of recombinant *E. coli* engineered bacteria containing a glufosinate dehydrogenase encoding gene and the amino acid sequence of the encoded protein are described in patent application CN202410819732.7.

[0037] As another implementation method, the crude enzyme solution or whole cells containing enzymes obtained by ultrasonically disrupting the bacterial cells for 10 minutes can also be used in the electrochemical-biological redox enzyme cascade catalysis method of the present invention.

[0038] Example 2: Screening and Preparation of Electrode Materials Based on the affinity of histidine-tagged enzyme proteins for metal ions and the principle of electrochemical coenzyme regeneration, platinum, titanium, nickel-molybdenum alloy, and copper were selected as working electrodes for the electrochemical reaction cell, respectively. These electrodes can promote oxidation reactions while simultaneously supporting coenzyme regeneration, providing NADH for the synthesis of L-2-aminobutyric acid and L-glufosinate. The preparation method of the working electrode for the electrochemical reaction cell is as follows: Solutions of threonine deaminase (TDH), leucine dehydrogenase (LeuDH), D-amino acid oxidase (DAAO), and glufosinate dehydrogenase (GHD) at a concentration of 0.1 mg / mL were prepared using Tris-HCl buffer (0.05 M, pH 8). A 3 cm × 3 cm × 0.1 cm metal electrode was placed in the enzyme solution and incubated at 4 °C with constant shaking at 180 rpm for 20 h. The electrode was then removed and placed at the working electrode position in the reactor.

[0039] Example 3: Using a platinum electrode as the anode and a titanium electrode as the cathode, a constant voltage of -0.5V was applied for the reaction. Prepare 100 mL of 50 mM D-2-amino-4-[hydroxy(methyl)phosphono]-butyric acid (D-PPT) using Tris-HCl buffer (0.05 M, pH 8), add DAAO enzyme and PpDH enzyme, and then add NAD to a final concentration of 5 mM. + The reactor was placed on a 50°C, 600 rpm electromagnetic stirring heater. A platinum electrode was clamped into the anode of the electrochemical workstation, and a titanium electrode into the cathode. The electrochemical workstation was set to change the polarity of the working electrodes every 10 minutes and output -0.5V for the reaction. Samples were taken every 0.5 hours and treated with derivatization reagents. A schematic diagram of the electrochemical-biosynthesis of L-glufosinate is shown below. Figure 1 As shown in the figure, the electrochemical-enzyme combination catalyzes the reaction of 50mM D-2-amino-4-[hydroxy(methyl)phosphono]-butyric acid (D-PPT) to L-2-amino-4-[hydroxy(methyl)phosphono]-butyric acid (L-PPT) at a voltage of -0.5V. Figure 2 As shown.

[0040] Fluorescence detection by high performance liquid chromatography (HPLC) (see...) Figure 2 The substrate residual concentration was 6.48 mM, the substrate conversion rate was 87.09%, the final L-PPT concentration was 87.13 mM, and the conversion rate was 86.35%.

[0041] Example 4: Using a platinum electrode as the anode and a titanium electrode as the cathode, a constant voltage of -0.6V was applied for the reaction. Prepare 100 mL of 50 mM D-2-amino-4-[hydroxy(methyl)phosphono]-butyric acid (D-PPT) using Tris-HCl buffer (0.05 M, pH 8), add DAAO enzyme and PpDH enzyme, and then add NAD to a final concentration of 5 mM. + The reactor was placed on a 50°C, 600 rpm electromagnetic stirrer. A platinum electrode was clamped into the anode of the electrochemical workstation, and a titanium electrode into the cathode. The electrochemical workstation was set to change the working electrode polarity every 10 minutes and output a -0.6V voltage for the reaction. Samples were taken every 0.5 hours and treated with derivatization reagents. The reaction process of 50 mM D-2-amino-4-[hydroxy(methyl)phosphono]-butyric acid (D-PPT) to L-2-amino-4-[hydroxy(methyl)phosphono]-butyric acid (L-PPT) catalyzed by the electrochemical-enzyme combination at -0.6V is shown in the figure below. Figure 3 As shown.

[0042] Fluorescence detection by high performance liquid chromatography (HPLC) (see...) Figure 3 The substrate residual concentration was 3.17 mM, the substrate conversion rate was 93.73%, and the final concentration of L-PPT after 2 h of reaction was 87.42 mM. The conversion rate of D-PPT to L-PPT was 86.62%.

[0043] Example 5: Using a platinum electrode as the anode and a titanium electrode as the cathode, a constant voltage of -0.7V was applied for the reaction. Prepare 100 mL of 50 mM D-2-amino-4-[hydroxy(methyl)phosphono]-butyric acid (D-PPT) using Tris-HCl buffer (0.05 M, pH 8), add DAAO enzyme and PpDH enzyme, and then add NAD to a final concentration of 5 mM. + The reactor was placed on a 50°C, 600 rpm electromagnetic stirrer. A platinum electrode was clamped into the anode of the electrochemical workstation, and a titanium electrode into the cathode. The electrochemical workstation was set to change the polarity of the working electrodes every 10 minutes and output a voltage of -0.7V for the reaction. Samples were taken every 0.5 hours and treated with derivatization reagents. The reaction process of 50 mM D-2-amino-4-[hydroxy(methyl)phosphono]-butyric acid (D-PPT) to L-2-amino-4-[hydroxy(methyl)phosphono]-butyric acid (L-PPT) catalyzed by the electrochemical-enzyme combination at -0.7V is shown in the figure below. Figure 4 As shown.

[0044] Fluorescence detection by high performance liquid chromatography (HPLC) (see...) Figure 4The substrate residual concentration was 1.53 mM, the substrate conversion rate was 96.67%, and after 2 h of reaction, the final concentration of L-PPT was 95.96 mM. The conversion rate of D-PPT to L-PPT was 94.52%. Using a voltage of -0.7 V as the reaction potential to couple DAAO enzyme and PpDH enzyme for catalytic reaction, the maximum conversion rate of the single potential catalytic system was achieved.

[0045] Example 6: Using a platinum electrode as the anode and a titanium electrode as the cathode, a constant voltage of -0.8V was applied for the reaction. Prepare 100 mL of 50 mM D-2-amino-4-[hydroxy(methyl)phosphono]-butyric acid (D-PPT) using Tris-HCl buffer (0.05 M, pH 8), add DAAO enzyme and PpDH enzyme, and then add NAD to a final concentration of 5 mM. + The reactor was placed on a 50°C, 600 rpm electromagnetic stirrer. A platinum electrode was clamped into the anode of the electrochemical workstation, and a titanium electrode into the cathode. The electrochemical workstation was set to change the working electrode polarity every 10 minutes and output a -0.8V voltage for the reaction. Samples were taken every 0.5 hours and treated with derivatization reagents. The reaction process of 50 mM D-2-amino-4-[hydroxy(methyl)phosphono]-butyric acid (D-PPT) to L-2-amino-4-[hydroxy(methyl)phosphono]-butyric acid (L-PPT) catalyzed by the electrochemical-enzyme combination at -0.8V is shown in the figure below. Figure 5 As shown.

[0046] Fluorescence detection by high performance liquid chromatography (HPLC) (see...) Figure 5 The substrate residual concentration was 1.78 mM, the substrate conversion rate was 96.47%, and the final concentration of L-PPT after 2 h of reaction was 94.20 mM. The conversion rate of D-PPT to L-PPT was 93.35%.

[0047] Example 7: Using a platinum electrode as the anode and a titanium electrode as the cathode, a constant voltage of -0.9V was applied for the reaction. Prepare 100 mL of 50 mM D-2-amino-4-[hydroxy(methyl)phosphono]-butyric acid (D-PPT) using Tris-HCl buffer (0.05 M, pH 8), add DAAO enzyme and PpDH enzyme, and then add NAD to a final concentration of 5 mM. +The reactor was placed on a 50°C, 600 rpm electromagnetic stirrer. A platinum electrode was clamped into the anode of the electrochemical workstation, and a titanium electrode into the cathode. The electrochemical workstation was set to change the working electrode polarity every 10 minutes and output a -0.9V voltage for the reaction. Samples were taken every 0.5 hours and treated with derivatization reagents. The reaction process of 50 mM D-2-amino-4-[hydroxy(methyl)phosphono]-butyric acid (D-PPT) to L-2-amino-4-[hydroxy(methyl)phosphono]-butyric acid (L-PPT) catalyzed by the electrochemical-enzyme combination at -0.9V is shown in the figure below. Figure 6 As shown.

[0048] Fluorescence detection by high performance liquid chromatography (HPLC) (see...) Figure 6 The substrate residual concentration was 2.69 mM, the substrate conversion rate was 94.65%, and the final concentration of L-PPT after 2 h of reaction was 93.20 mM, with a conversion rate of 92.20% from D-PPT to L-PPT.

[0049] Example 8: Catalytic experiments were conducted using a platinum electrode as the anode and a titanium electrode as the cathode, employing a multi-segment pulse cycle with different periods. Prepare 100 mL of 50 mM D-2-amino-4-[hydroxy(methyl)phosphono]-butyric acid (D-PPT) using Tris-HCl buffer (0.05 M, pH 8), add DAAO enzyme and PpDH enzyme, and then add NAD to a final concentration of 5 mM. + The reactor was placed on an electromagnetic stirrer at 50°C and 600 rpm. The platinum electrode was clamped into the anode of the electrochemical workstation, and the titanium electrode was clamped into the cathode. The single voltage energizing time was set to 0.5 minutes (i.e., initially setting Pt as the working electrode and continuously applying a voltage of -0.7V for 0.5 minutes, then switching to -0.1V for 0.5 minutes, then setting the electrode parameter to Ti electrode and continuously applying a voltage of -0.8V for 0.5 minutes, which constitutes one cycle, and then entering the next cycle. The reaction was repeated for 2 hours, during which the hydrogen peroxide decomposition potential of -0.1V was set to energize for 0.5 minutes in 6 different parameter cycles), 1 minute, 2 minutes, 3 minutes, 5 minutes, and 10 minutes. The reaction process of electrochemical-enzyme combination catalyzing the conversion of 50mM D-2-amino-4-[hydroxy(methyl)phosphono]-butyric acid (D-PPT) to L-2-amino-4-[hydroxy(methyl)phosphono]-butyric acid (L-PPT) after the introduction of pulse potentials with six different periods is shown in the figure below. Figure 7 As shown.

[0050] Experimental results are as follows Figure 7As shown, the conversion rates at 2 hours under six different cycles were 88.6%, 90.1%, 91.6%, 91.0%, 89.6%, and 89.2%, respectively. With the extension of the single-cycle energizing time, the catalytic efficiency initially increased and then decreased. This phenomenon is mainly due to the following: when the energizing time is too short within a single cycle, D-PPT cannot be converted into the intermediate product in time, and the electrochemical regeneration of NADH is not optimal, failing to convert the intermediate product PPO into L-PPT in time. With the extension of the single-cycle energizing time, D-PPT can be better converted, and NADH can be effectively regenerated. However, when the single-cycle energizing time is too long, D-PPT is again converted into the intermediate product PPO, and the necessary NADH for PpDH enzyme is not provided electrochemically in time. Furthermore, PPO is easily decomposed, leading to a decrease in conversion rate.

[0051] Example 9: Using a platinum electrode as the anode and a titanium electrode as the cathode, the reaction concentration was increased, and a multi-segment pulsed potential reaction was employed. Prepare 100 mL of 200 mM D-2-amino-4-[hydroxy(methyl)phosphono]-butyric acid (D-PPT) using Tris-HCl buffer (0.05 M, pH 8), add DAAO enzyme and PpDH enzyme, and then add NAD to a final concentration of 15 mM. + The reactor was placed on a 50℃, 600 rpm electromagnetic stirrer. A platinum electrode was clamped to the anode of the electrochemical workstation, and a titanium electrode to the cathode. A potential of -0.7V, favorable for oxidation, was applied for electro-enzyme synergistic catalysis for 2 minutes. Then, a potential of -0.2V, favorable for hydrogen peroxide decomposition, was applied for 1 minute of catalytic reaction. Finally, a potential of -0.8V, favorable for reduction, was applied for 2 minutes of catalytic reaction. This was repeated as one pulse cycle for potential cycling. The total catalytic time was set to 8 hours, with samples taken every 2 hours and treated with derivatization reagents. The reaction process of catalyzing the conversion of 50 mM D-2-amino-4-[hydroxy(methyl)phosphono]-butyric acid (D-PPT) to L-2-amino-4-[hydroxy(methyl)phosphono]-butyric acid (L-PPT) after multi-segment pulsed potential reaction is shown in the figure. Figure 8 As shown.

[0052] Figure 8 Experimental results show that fluorescence detection by high-performance liquid chromatography (HPLC) (see...) Figure 8The substrate residual concentration was 16.79 mM, the substrate conversion rate was 91.62%, and the final L-PPT concentration was 384.16 mM after 8 h of reaction. Finally, using multi-segment pulse potential coupling of DAAO enzyme and PpDH enzyme in the absence of biological coenzyme regeneration system, the conversion rate of D-PPT to L-PPT was 95.62%.

[0053] Example 10: L-2-aminobutyric acid was synthesized by coupling threonine deaminase and leucine dehydrogenase with four metal electrodes (platinum, titanium, nickel-molybdenum alloy, and copper) and applying the optimal reaction potential to each electrode. Prepare four 100 mL aliquots of 100 mM threonine solution using Tris-HCl buffer (0.05 M, pH 8), add TDH enzyme and LeuDH enzyme, and then add NAD+ to a final concentration of 15 mM. + The reactor was placed on a 35°C, 600 rpm electromagnetic stirring heater. Four metal electrodes (platinum, titanium, nickel-molybdenum alloy, and copper) were clamped into the working electrode of the electrochemical workstation, and a graphite electrode was clamped into the auxiliary electrode. The electrochemical workstation was set to change the polarity of the working electrode every 10 minutes, and the four reactors were operated at their respective optimal reaction potentials (-1.4V, -0.8V, -1.4V, and -0.8V). Samples were taken every 2 hours and treated with derivatization reagents. The efficiency of L-2-aminobutyric acid production using four different metal electrodes under their respective optimal catalytic potentials is shown in the figure below. Figure 9 As shown.

[0054] Figure 9 Experimental results show that fluorescence detection by high-performance liquid chromatography (HPLC) (see...) Figure 9 Four metal electrodes coupled with TDH and LeuDH enzymes were used to synthesize L-2-threonine. Among them, the Ti electrode as the working electrode achieved the best conversion rate of 93.37% at a potential of -0.8V, while the Cu electrode as the working electrode had the weakest effect, with a conversion rate of only 72.58% at a potential of -0.8V. The conversion rates of Pt electrode and Ni-Mo alloy as working electrodes at a potential of -1.4V were 78.11% and 87.17%, respectively.

[0055] The above description is merely a detailed explanation of preferred embodiments and principles of the present invention. For those skilled in the art, there may be changes in specific implementation methods based on the ideas provided by the present invention, and these changes should also be considered within the scope of protection of the present invention.

Claims

1. An electrochemical-biological redox enzyme cascade catalytic method, characterized in that, Includes the following steps: In an electrochemical reaction cell, an electrochemical workstation is used to control the polarity of the working electrode, causing it to alternate between the anode and cathode to promote the catalytic reaction of at least one redox enzyme and achieve the electrochemical regeneration of the coenzyme; wherein the polarity switching of the working electrode is achieved by potential adjustment, thereby synthesizing the target compound.

2. The electrochemical-biological redox enzyme cascade catalytic method according to claim 1, characterized in that, The oxidoreductase is selected from at least one of dehydrogenase and oxidase.

3. The electrochemical-biological redox enzyme cascade catalytic method according to claim 2, characterized in that, The dehydrogenase is any one or a combination of two of leucine dehydrogenase and glufosinate dehydrogenase; the oxidase is any one or a combination of two of D-amino acid oxidase and threonine deaminase.

4. The electrochemical-biological redox enzyme cascade catalytic method according to claim 1, characterized in that, The working electrode is made of any one or more of the following materials: platinum, titanium, nickel-molybdenum alloy, copper, graphite, and gold.

5. The electrochemical-biological redox enzyme cascade catalytic method according to claim 1, characterized in that, The potential adjustment employs a multi-segment pulsed reaction potential, which includes a combination of at least two of the following: oxidation potential, reduction potential, and hydrogen peroxide decomposition potential.

6. The electrochemical-biological redox enzyme cascade catalytic method according to claim 5, characterized in that, The cycle period of the pulse voltage of the multi-segment pulsed reactive potential is 0.5~10 min.

7. The electrochemical-biological redox enzyme cascade catalytic method according to claim 1, characterized in that, The coenzyme is NAD. + / NADH, the regeneration of the coenzyme is achieved through cathodic reduction.

8. The electrochemical-biological redox enzyme cascade catalytic method according to claim 1, characterized in that, The reaction medium in the electrochemical reaction cell is a buffer solution with a pH of 7-9.

9. The electrochemical-biological redox enzyme cascade catalytic method according to claim 1, characterized in that, The catalytic reaction is carried out at a temperature of 35-50°C, a pressure of 1.01 MPa, and a time of 0.5-8 h.

10. The electrochemical-biological redox enzyme cascade catalytic method according to claim 1, characterized in that, The target compound is a chiral amino acid or a derivative thereof.

Images