Co-immobilized multienzyme catalyst, preparation method thereof and application of co-immobilized multienzyme catalyst in carbon dioxide conversion
By fixing carbonate enzyme enzymes and methal more hydrogenase with glutamic acid dehydrinase, combined into a polyase catalyst, solving the problem of CO2 texture and constitutional restrictions in polyase -level liquid catalysis, realized high efficiency, low cost The effect of carbon dioxide into methic acid.
Patent Information
- Application Number
- CN202411453937.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-10-17
- Publication Date
- 2025-01-24
AI Technical Summary
During the catalytic conversion of carbon dioxide by multi-enzyme cascade, there are problems such as gas-liquid mass transfer limitation of CO2, conformational limitation caused by co-fixation of multiple enzymes, and low efficiency of coupling and coenzyme regeneration, which limits the carbon dioxide to high value-added chemicals Effective conversion.
By immobilizing carbonic anhydrase (CA) on UiO materials, and co-immobilizing formate dehydrogenase (FDH) and coenzyme glutamate dehydrogenase (GDH) on ZIF materials, a co-immobilized multi-enzyme catalyst is formed. Realize the regeneration of coenzymes and efficient catalytic conversion of CO2 into formic acid.
This method effectively solves the problems of CO2 gas-liquid mass transfer limitations and multi-enzyme co-fixed conformation limitations, reduces costs through coenzyme regeneration, and achieves the effect of efficiently catalyzing the conversion of CO2 into formic acid.
Smart Images
Figure HDA0005089926250000011 
Figure HDA0005089926250000012 
Figure HDA0005089926250000021
Abstract
Description
Technical Field
[0001] The invention belongs to the field of carbon dioxide recovery and conversion, and specifically relates to a co-immobilized multi-enzyme catalyst and a preparation method thereof and application thereof in carbon dioxide conversion. Background Art
[0002] The climate crisis caused by excessive carbon dioxide emissions has attracted widespread attention from the international community. To this end, countries have gradually carried out many effective actions. At present, the development of artificial multi-enzyme cascade reaction pathways to produce high-value C using CO2 as raw material is 2+ Compounds or their reduced C1 products (such as formic acid) are an emerging hot topic in the field of biomanufacturing. The multi-enzyme cascade system based on carbonic anhydrase and formate dehydrogenase for catalytic conversion of CO2 to stable one-carbon formate intermediates has attracted widespread attention from researchers. It is worth noting that the participation of coenzymes is often required in the formate dehydrogenase reaction process, but the high cost of coenzymes poses a challenge. Therefore, the development of immobilized multi-enzyme carriers that can be coupled to the coenzyme regeneration link and the cascade catalytic conversion of CO2 is an efficient, sustainable and highly valuable immobilized enzyme strategy.
[0003] At present, in the process of multi-enzyme cascade catalytic conversion of carbon dioxide, there are still problems such as gas-liquid mass transfer limitation of CO2, conformational limitation caused by multi-enzyme co-immobilization, and low efficiency of coupled coenzyme regeneration, which restrict the conversion of CO2 into high value-added chemicals in practical applications.
[0004] Carbonic anhydrase (CA) and formate dehydrogenase (FDH) are two key biocatalysts for this conversion, which promote the catalytic conversion of CO2 to formic acid in a complementary manner. Carbonic anhydrase can promote the conversion of carbon dioxide to HCO3 - FDH is a redox enzyme that can specifically and reversibly catalyze the reduction of CO2 to formic acid. However, the catalysis of formate dehydrogenase usually requires the participation of reduced nicotinamide adenine dinucleotide (NADH), and its expensive cost has limited its practical application. For this reason, researchers have reported that by introducing glutamate dehydrogenase into the reaction system, NADH can be regenerated, thereby reducing the cost of catalytic conversion of CO2 to formic acid. In addition, the instability of the free enzyme itself and the difficulty of recovering it from the reaction system are another important factor limiting this reaction. Therefore, it is of great significance to develop an enzyme system that efficiently catalyzes the conversion of CO2 to formic acid. Summary of the invention
[0005] The technical problem to be solved by the present invention is to provide a co-immobilized multi-enzyme catalyst, a preparation method thereof and an application in carbon dioxide conversion. The co-immobilized multi-enzyme catalyst co-immobilizes glutamate dehydrogenase (GDH) with CA and FDH, can achieve the regeneration of coenzymes, reduce costs and achieve efficient catalytic conversion of CO2 to formic acid.
[0006] The technical solution of the present invention is as follows:
[0007] A method for preparing a co-immobilized multi-enzyme catalyst comprises the following steps:
[0008] (1) Immobilizing carbonic anhydrase (CA) on UiO material to obtain immobilized carbonic anhydrase;
[0009] (2) immobilizing formate dehydrogenase (FDH) and coenzyme glutamate dehydrogenase (GDH) on ZIF material to obtain immobilized dehydrogenase;
[0010] (3) The immobilized carbonic anhydrase obtained in step (1) and the immobilized dehydrogenase obtained in step (2) are mixed and shaken to obtain the co-immobilized multi-enzyme catalyst.
[0011] Preferably, in step (1), the UiO material is DUiO-67.
[0012] DUiO-67 is a defective body of UiO-67, and the preparation method is as follows:
[0013] Dissolve ZrCl4 and biphenyl-4,4'-dicarboxylic acid in N,N-dimethylamide, add acetic acid and stir at 20-30°C for 1-5h, then transfer the mixed solution into a hydrothermal reactor, further add DMF, and react in a preheated oven at 120-130°C for 20-30h. After the reaction is completed, collect by centrifugation to obtain DUiO-67
[0014] Preferably, in step (1), the mass ratio of the UiO material to the carbonic anhydrase is 5:0.25-3.
[0015] Preferably, in step (1), the method for immobilizing carbonic anhydrase on the UiO material is as follows:
[0016] The UiO material is dispersed in a Tris-HCl buffer, and then a carbonic anhydrase solution is added, and the reaction is carried out at 20-30° C. and 100-200 rpm for 2-10 hours. The solid is collected, and then dispersed in a P3 primer, and then allowed to stand for aging to obtain immobilized carbonic anhydrase.
[0017] Preferably, in step (2), the ZIF material is ZIF-L or ZIF-8, wherein the ZIF-L material is leaf-shaped.
[0018] Preferably, in step (2), the preparation method of the immobilized dehydrogenase is as follows:
[0019] Formate dehydrogenase, glutamate dehydrogenase, streptavidin and zinc nitrate hexahydrate aqueous solution were mixed, and then 2-methylimidazole aqueous solution was added, and stirred for reaction. After the reaction was completed, the precipitate was collected and dispersed into the P1 primer, and the stirring reaction was continued to obtain the immobilized dehydrogenase.
[0020] Furthermore, in terms of mass, ZnNO 3 : 2-methylimidazole : formate dehydrogenase : glutamate dehydrogenase = 29.6 : 65.5 : 0.1 to 7 : 0.1 to 7, and more preferably 29.6 : 65.5 : 6 : 6.
[0021] The present invention also provides a co-immobilized multi-enzyme catalyst obtained by the above preparation method.
[0022] The present invention also provides a method for preparing formic acid by using CO2. In L-glutamic acid, NADH and PBS buffer, CO2 reacts under the action of the above-mentioned co-immobilized multi-enzyme catalyst to obtain formic acid.
[0023] Preferably, in the above preparation method, the L-glutamic acid, NADH and PBS buffer are first prepared into a substrate solution, CO2 is then introduced for 3 to 10 minutes, the co-immobilized multi-enzyme catalyst is added, and finally CO2 is continued to be introduced for reaction to obtain the formic acid.
[0024] Compared with the prior art, the beneficial effects of the present invention are as follows:
[0025] The present invention solves the gas-liquid mass transfer problem of CO2 and the problem of low enzyme loading in MOFs by fixing carbonic anhydrase (CA) on the surface of UiO material with mesopores; then formate dehydrogenase (FDH) and coenzyme regeneration enzyme-glutamate dehydrogenase (GDH) are co-fixed in a blade-type ZIF with a large specific surface area, thereby improving the efficiency of coenzyme regeneration; finally, the above two immobilized enzyme modules are combined together to realize compartmental fixation, thereby solving the problem of conformational restriction caused by co-immobilization of multiple enzymes and avoiding the risk of failure of the entire immobilization system due to inactivation of a single enzyme. BRIEF DESCRIPTION OF THE DRAWINGS
[0026] Figure 1The SEM diagrams of the immobilized multi-enzyme catalyst obtained in Example 1 and the intermediates of each step, wherein (a) to (f) represent the following structures: DUiO-67@P3 (a), ZIF-L@P1 (b), DUiO-67@P3@ZIF-L@P1 (c), CA@DUiO-67@P3 (d), FDH / GDH@ZIF-L@P1 (e) and CA@DUiO-67@P3@FDH / GDH@ZIF-L@P1 (f);
[0027] Figure 2 TEM (ac) and TEM-mapping (d) images of the immobilized multi-enzyme catalyst 1 obtained in Example 1;
[0028] Figure 3 is the CLSM image of the immobilized multi-enzyme catalyst 1 obtained in Example 1;
[0029] Figure 4 The yield graph of formic acid obtained in Test Examples 1 to 5;
[0030] Figure 5 The yield graph of formic acid obtained in Test Examples 1, 6 to 9;
[0031] Figure 6 is the stability test result of test example 10;
[0032] Figure 7 This is a graph comparing the formic acid production of each multi-enzyme system at different reaction times in Test Example 1. DETAILED DESCRIPTION
[0033] The present invention is further described below through specific examples.
[0034] Unless otherwise specified, the reagents and materials used in the examples are commercially available products.
[0035] The preparation method of DUiO-67 is as follows:
[0036] ZrCl4 (0.4 mM) and biphenyl-4,4'-dicarboxylic acid (BPDC) (0.4 mM) were dissolved in 30.85 mL N,N-dimethylformamide (DMF), 10 mM acetic acid was added and stirred at room temperature for 2 h. The mixed solution was transferred into a hydrothermal reactor with a Teflon liner, and 10 mL DMF was added and reacted in a preheated 120 ° C oven for 24 h. After the reaction was completed, DUiO-67 was collected by centrifugation.
[0037] DUiO-67 obtained by this method is a defective MOF with larger pores.
[0038] P1 and P3 are primers commonly used in genetic engineering. The sequences of P1 and P3 in the present invention are as follows:
[0039] P1: 5′-Biotin-CCGCCATTAAGGTGGCCTAAGGGCCGGGCCAAAT TTTTTAA-3′.
[0040] P3: 5′-NH2-C 2～10 -TTAAAAAATTTTGGCCCGGCCCTTAGGCCACCT TAATGGCGG-3′.
[0041] Example 1
[0042] (1) Preparation of CA@DUiO-67
[0043] 50 mg of DUiO-67 was dispersed in 9.5 mL of Tris-HCl buffer (50 mM, pH = 6.0), and then 500 μL of CA enzyme solution (40 mg / mL, Milli-Q) was added. The mixture was placed in a shaking incubator and reacted at 25 ° C and 150 rpm for 3 h. The solid CA@DUiO-67 was collected by centrifugation.
[0044] (2) Preparation of CA@DUiO-67@P3
[0045] The prepared 40 mg CA@DUiO-67 was mixed with 100 μL P3 aqueous solution (50 μM) and aged for 2 h to obtain CA@DUiO-67@P3.
[0046] (3) Preparation of FDH / GDH@ZIF-L@P1
[0047] 6 mg of formate dehydrogenase (FDH) and 6 mg of glutamate dehydrogenase (GDH) were pre-mixed with 120 μL of 5 mg / mL streptavidin (SA) and 2 mL of zinc nitrate hexahydrate aqueous solution (29.6 mg) for 15 min, and 2 mL of 2-methylimidazole aqueous solution (65.6 mg) was added. Then, the reaction mixture was reacted at 30 ° C and 200 rpm for 4 h. The collected precipitate was then redispersed in P1 (0.1 mM, 411.4 μL) aqueous solution and stirred for 2 h. FDH / GDH@ZIF-L@P1 (denoted as FDH&GDH / ZIF=3:1) was obtained.
[0048] (4) Preparation of CA@DUiO-67@P3@FDH / GDH@ZIF-L@P1
[0049] 20 mg of CA@DUiO-67@P3 and 20 mg of FDH / GDH@ZIF-L@P1 were weighed into 1 mL of ultrapure water. The mixed solution was then placed in an oscillating incubator and oscillated at 18°C and 150 rpm for 2 h to obtain the co-immobilized multi-enzyme catalyst 1 (ZIF-L / DUiO-67 = 1:1). The solution was stored at 4°C for subsequent use.
[0050] The SEM images of the immobilized multi-enzyme catalyst 1 obtained in this example and the intermediates of each step are shown in Figure 1 . Figure 1 The results show that after coating P3 on the surface of CA@DUiO-67 ( Figure 1 (d)), its morphology and particle size have no obvious changes compared with DUiO-67@P3. FDH / GDH@ZIF-L@P1( Figure 1 (e)) has the same surface morphology and size as FDH / GDH@ZIF-L. Figure 1 (f) It can be observed that CA@DUiO-67@P3 with regular polyhedral morphology is dispersed on the surface of FDH / GDH@ZIF-L@P1, indicating the successful preparation of the self-assembly immobilized multi-enzyme system (CA@DUiO-67@P3@FDH / GDH@ZIF-L@P1).
[0051] Figure 2 TEM and TEM-mapping images of the immobilized multi-enzyme catalyst 1 or intermediate obtained in Example 1. Figure 2 It can be seen that ZIF-L crystals present a typical leaf-like morphology ( Figure 2 (a) and Figure 2 (b)), whose size is basically consistent with the SEM results ( Figure 1 (c)). A large number of DUiO-67@P3 crystals are distributed on the surface of ZIF-L@P1, indicating the successful preparation of the self-assembly composite carrier (DUiO-67@P3@ZIF-L@P1). However, the local magnified TEM image ( Figure 2 (c)) reveals that some DUiO-67@P3 crystals have aggregated and are not evenly distributed on the ZIF-L@P1 surface.
[0052] TEM-mapping results ( Figure 2 (eg)) further confirmed the above findings. The distribution of Zn element ( Figure 2 (f)) are leaf-shaped and evenly and densely distributed, which is different from the TEM morphology of ZIF-L ( Figure 2 (d)). In addition, the Zr element ( Figure 2(g)) are dispersed in the ZIF-L region and its edge where the Zn element exists. This finding further proves the successful preparation of the composite carrier by self-assembly. It is worth noting that the Zr element is not evenly distributed, especially in the right area of the crystal ( Figure 2 (e)) presents a larger density, which is consistent with the distribution characteristics of DUiO-67 on the composite support observed by SEM.
[0053] FITC, AMC, and Rhd-B were used as fluorescent markers to label CA, GDH, and FDH. By collecting the corresponding fluorescence signals at different excitation wavelengths, we observed the distribution of multi-enzymes in the self-assembly immobilized multi-enzyme system. The experimental results are shown in Figure 3 .
[0054] like Figure 3 As shown in (ac), green, blue and red fluorescence signals generated by FITC-CA, AMC-GDH and Rhd-B-FDH can be observed respectively, which confirms the successful labeling of the three enzymes by fluorescent dyes. The fluorescence signals of the three enzymes are evenly distributed and have high fluorescence intensity, which indicates the successful preparation of the self-assembly method for immobilizing multi-enzyme system.
[0055] CLSM images of the self-assembly immobilized multi-enzyme system ( Figure 3 In (d), we observed purple-red fluorescence, which is the result of a mixture of blue and red fluorescence, indicating that FDH and GDH have been successfully co-immobilized in ZIF-L. In addition, we also observed a small amount of pink fluorescence, which is the product of a mixture of red, blue and green fluorescence. Furthermore, in some areas, the green fluorescence signal of CA is more significant, while the red / blue signals of FDH and GDH are relatively weak. In addition, the green fluorescence signal of CA does not completely overlap with the fluorescence signals of FDH and GDH. This phenomenon reveals that the CA enzyme is not immobilized on the same carrier as FDH and GDH. From the fluorescence signal intensity and area, it can be inferred that the three labeled enzymes have been successfully immobilized on the expected carrier. Since the self-assembly method is assembled after the preparation of the two immobilized enzymes, the integrity of the original immobilized enzymes is ensured.
[0056] From the analysis of the fluorescence signal intensity, the fluorescence intensity of CA was slightly weaker than that of FDH and GDH, which may suggest that the immobilization amount of CA enzyme was lower than that of FDH and GDH.
[0057] Example 2
[0058] The operating conditions of Example 2 are similar to those of Example 1, except that in step (4), the amounts of CA@DUiO-67@P3 and FDH / GDH@ZIF-L@P1 used are 30 mg and 10 mg, respectively, to obtain a co-immobilized multi-enzyme catalyst 2 (ZIF-L / DUiO-67=1:3).
[0059] Example 3
[0060] The operating conditions of Example 3 are similar to those of Example 1, except that in step (4), the amounts of CA@DUiO-67@P3 and FDH / GDH@ZIF-L@P1 used are 20 mg and 10 mg, respectively, to obtain a co-immobilized multi-enzyme catalyst 3 (ZIF-L / DUiO-67=1:2).
[0061] Example 4
[0062] The operating conditions of Example 4 are similar to those of Example 1, except that in step (4), the amounts of CA@DUiO-67@P3 and FDH / GDH@ZIF-L@P1 used are 10 mg and 30 mg, respectively, to obtain a co-immobilized multi-enzyme catalyst 4 (ZIF-L / DUiO-67=3:1).
[0063] Example 5
[0064] The operating conditions of Example 5 are similar to those of Example 1, except that in step (4), the amounts of CA@DUiO-67@P3 and FDH / GDH@ZIF-L@P1 used are 10 mg and 20 mg, respectively, to obtain a co-immobilized multi-enzyme catalyst 5 (ZIF-L / DUiO-67=2:1).
[0065] Test cases 1 to 5
[0066] 10 mM L-glutamic acid and 10 mM NADH were dissolved in 10 mL PBS buffer (50 mM, pH = 7.0) to prepare a substrate solution. CO2 was then blown into the substrate solution at a constant flow rate for 5 min, and then 15 mg of CA@DUiO-67@P3@FDH / GDH@ZIF-L@P1 obtained in Examples 1 to 5 was added to the substrate solution, and carbon dioxide was continued to be blown for 2.5 hours. After passing through the membrane, the formic acid production was determined by high performance liquid chromatography. The results are shown in Figure 4 .
[0067] Depend on Figure 4 It can be seen that the catalytic efficiency of the co-immobilized multi-enzyme catalyst 1 (ZIF-L / DUiO-67=1:1) was the highest, reaching 21.30 mM (2.5 h).
[0068] Example 6
[0069] The operating conditions of Example 6 are similar to those of Example 1, except that in step (3), the formate dehydrogenase (FDH) and glutamate dehydrogenase (GDH) are each 4 mg, and a co-immobilized multi-enzyme catalyst 6 is obtained (FDH&GDH / ZIF=2:1).
[0070] Example 7
[0071] The operating conditions of Example 7 are similar to those of Example 1, except that in step (3), the formate dehydrogenase (FDH) and glutamate dehydrogenase (GDH) are each 2 mg, and a co-immobilized multi-enzyme catalyst 7 is obtained (FDH&GDH / ZIF=1:1).
[0072] Example 8
[0073] The operating conditions of Example 8 are similar to those of Example 1, except that in step (3), the formate dehydrogenase (FDH) and glutamate dehydrogenase (GDH) are each 1 mg, and a co-immobilized multi-enzyme catalyst 8 is obtained (FDH&GDH / ZIF=1:2).
[0074] Example 9
[0075] The operating conditions of Example 9 are similar to those of Example 1, except that in step (3), the amounts of formate dehydrogenase (FDH) and glutamate dehydrogenase (GDH) are each 0.67 mg, to obtain a co-immobilized multi-enzyme catalyst 9 (denoted as FDH&GDH / ZIF=1:3).
[0076] Test cases 6 to 9
[0077] 10 mM L-glutamic acid and 10 mM NADH were dissolved in 10 mL PBS buffer (50 mM, pH = 7.0) to prepare a substrate solution. CO2 was then blown into the substrate solution at a constant flow rate for 5 min, and then 15 mg of CA@DUiO-67@P3@FDH / GDH@ZIF-L@P1 obtained in Examples 6 to 9 was added to the substrate solution. Carbon dioxide was continued to be blown for 2.5 hours to obtain formic acid. The yield of formic acid was measured. The results are shown in Figure 5 .
[0078] Test Example 10: Stability Test
[0079] After the reaction of Test Example 1 was completed, the co-immobilized multi-enzyme catalyst 1 was centrifuged and collected. 10mM L-glutamic acid and 10mM NADH were dissolved in 10mL PBS buffer (50mM, pH=7.0) to prepare a new substrate solution. Then, CO2 was blown into the substrate solution at a constant flow rate for 5min, and the immobilized enzyme collected by centrifugation was added. Carbon dioxide was continued to be blown for 2h. After passing through the membrane, the formic acid production was determined by high performance liquid chromatography. The determination was repeated 10 times. The results are shown in FIG. Figure 6 .
[0080] The results of this test example show that the co-immobilized multi-enzyme catalyst 1 obtained in the present invention has high stability.
[0081] Comparative Example 1
[0082] 6 mg of formate dehydrogenase (FDH) and 6 mg of glutamate dehydrogenase (GDH) were pre-mixed with 2 mL of zinc nitrate hexahydrate aqueous solution (29.6 mg) for 15 min, and 2 mL of 2-methylimidazole aqueous solution (65.6 mg) was added. Then, the reaction mixture was reacted at 30 ° C and 200 rpm for 4 h to obtain the FDH / GDH@ZIF-L dual enzyme system.
[0083] Comparative test example 1
[0084] 10 mM L-glutamic acid and 10 mM NADH were dissolved in 10 mL PBS buffer (50 mM, pH = 7.0) to prepare a substrate solution. CO2 was then blown into the substrate solution at a constant flow rate for 5 min, and then 115 mg of CA@DUiO-67@P3@FDH / GDH@ZIF-L@P1 obtained in Example 1 and the FDH / GDH@ZIF-L dual enzyme system obtained in Comparative Example 1 were added to the substrate solution, and carbon dioxide was continued to be blown. After passing through the membrane every half an hour, the formic acid production was determined by high performance liquid chromatography to obtain formic acid. The results are shown in FIG. Figure 7 .
[0085] Figure 7 The results showed that the formic acid production of the immobilized FDH / GDH@ZIF-L dual-enzyme system was much lower than that of the immobilized multi-enzyme system, indicating that the ability of FDH itself to participate in the catalytic conversion of CO2 was lower than the ability of the cascade catalytic conversion of CA and FDH.
Claims
1. A method for preparing a co-immobilized multi-enzyme catalyst, characterized in that: The following steps are involved: (1) Immobilizing carbonic anhydrase (CA) on UiO material to obtain immobilized carbonic anhydrase; (2) immobilizing formate dehydrogenase (FDH) and coenzyme glutamate dehydrogenase (GDH) on ZIF material to obtain immobilized dehydrogenase; (3) The immobilized carbonic anhydrase obtained in step (1) and the immobilized dehydrogenase obtained in step (2) are mixed and shaken to obtain the co-immobilized multi-enzyme catalyst.
2. The method for preparing the co-immobilized multi-enzyme catalyst according to claim 1, characterized in that: In step (1), the UiO material is DUiO-67.
3. The method for preparing the co-immobilized multi-enzyme catalyst according to claim 1, characterized in that: In step (1), the mass ratio of the UiO material to the carbonic anhydrase is 5:0.25-3.
4. The method for preparing the co-immobilized multi-enzyme catalyst according to claim 1, characterized in that: In step (1), the method for immobilizing carbonic anhydrase on the UiO material is as follows: The UiO material is dispersed in a Tris-HCl buffer, and then a carbonic anhydrase solution is added, and the reaction is carried out at 20-30° C. and 100-200 rpm for 2-10 hours. The solid is collected, and then dispersed in a P3 primer, and then allowed to stand for aging to obtain immobilized carbonic anhydrase.
5. The method for preparing the co-immobilized multi-enzyme catalyst according to claim 1, characterized in that: In step (2), the ZIF material is ZIF-L or ZIF-8.
6. The method for preparing the co-immobilized multi-enzyme catalyst according to claim 1, characterized in that: In step (2), the preparation method of immobilized dehydrogenase is as follows: Formate dehydrogenase, glutamate dehydrogenase, streptavidin and zinc nitrate hexahydrate aqueous solution were mixed, and then 2-methylimidazole aqueous solution was added, and stirred for reaction. After the reaction was completed, the precipitate was collected and dispersed into the P1 primer, and the stirring reaction was continued to obtain the immobilized dehydrogenase.
7. A co-immobilized multi-enzyme catalyst obtained by the preparation method according to any one of claims 1 to 6.
8. A method for preparing formic acid using CO2, characterized in that: In L-glutamic acid, NADH and PBS buffer, CO2 reacts under the action of the co-immobilized multi-enzyme catalyst of claim 7 to obtain formic acid.
9. The method for preparing formic acid using CO2 according to claim 8, characterized in that: First, the L-glutamic acid, NADH and PBS buffer are prepared into a substrate solution, then CO2 is introduced for 3 to 10 minutes, and then the co-immobilized multi-enzyme catalyst is added, and finally CO2 is continued to be introduced for reaction to obtain the formic acid.