Enzyme-containing catalyst, preparation method thereof and application of enzyme-containing catalyst in preparation of L-lactic acid

Abstract

The invention relates to the technical field of catalysts, in particular to an enzyme-containing catalyst, a preparation method thereof and application of the enzyme-containing catalyst to preparation of L-lactic acid. The preparation method of the enzyme-containing catalyst comprises the following steps: adding Co-TCPP, L-lactic dehydrogenase and pyruvate decarboxylase into a 2-methylimidazole aqueous solution, fully dissolving, dropwise adding a zinc nitrate hexahydrate solution and a polyvinylpyrrolidone solution, stirring to generate a precipitate, and washing and drying to obtain the enzyme-containing catalyst. Filling CO2 into the buffer solution until the buffer solution is saturated, adding acetaldehyde, an enzyme-containing catalyst and oxidized coenzyme NAD < + >, providing an illumination condition, and converting into L-lactic acid by taking CO2 as a raw material. According to the method, Co-TCPP is compounded through the synthesized enzyme-containing catalyst, on the basis of large-aperture MOF loaded enzyme, the L-lactic acid is synthesized by coupling the synthesized enzyme-containing catalyst with an auxiliary factor for regeneration and taking CO2 as a raw material, and a high catalytic effect can be obtained under very mild conditions.

Description

Technical Field

[0001] The invention relates to the technical field of catalysts, and in particular to an enzyme-containing catalyst, a preparation method thereof, and application of the catalyst in preparing L-lactic acid. Background Art

[0002] A multi-enzyme system for producing L-lactic acid from CO2 and acetaldehyde, which consists of two enzymes: pyruvate decarboxylase (PyDc) converts CO2 and acetaldehyde into pyruvate, and lactate dehydrogenase (LDH) converts pyruvate into L-lactic acid consuming NADH. However, there are some urgent problems to be solved in reducing CO2 to produce L-lactic acid: (1) The photoactivity of the catalyst is poor; (2) Expensive electron mediator M is required to improve the yield, resulting in increased reaction costs; (3) The reaction rate is slow and requires catalysis under long-term light conditions. Summary of the invention

[0003] The purpose of the present invention is to address the above-mentioned deficiencies in the prior art and to provide an enzyme-containing catalyst and a preparation method thereof and an application thereof in the preparation of L-lactic acid.

[0004] The invention discloses a method for preparing an enzyme-containing catalyst. The method comprises the following steps: adding Co-TCPP, L-lactate dehydrogenase and pyruvate decarboxylase into a 2-methylimidazole aqueous solution, and after fully dissolving, adding a zinc nitrate hexahydrate solution and a polyvinyl pyrrolidone solution dropwise, stirring to generate a precipitate, washing and drying to obtain the enzyme-containing catalyst PyDc&LDH&Co-TCPP@ZIF-8.

[0005] Furthermore, the mass of Co-TCPP is 0.1%-5% of the mass of the generated ZIF-8.

[0006] Furthermore, the molar mass ratio of zinc nitrate hexahydrate to L-lactate dehydrogenase and pyruvate decarboxylase is 0.2-0.35 mmol: 5-7 mg: 5-7 mg.

[0007] Furthermore, L-lactate dehydrogenase and pyruvate decarboxylase are added as enzyme solutions, and the concentrations of the two enzyme solutions are 1-5 mg / mL.

[0008] An enzyme-containing catalyst prepared by the above preparation method.

[0009] An application of the enzyme-containing catalyst as described above in the preparation of L-lactic acid is to charge CO2 into a buffer until it is saturated, and then add acetaldehyde, the enzyme-containing catalyst and the oxidized coenzyme NAD + , provide light conditions, and convert CO2 as raw material into L-lactic acid.

[0010] Furthermore, the buffer is a phosphate buffer with a pH of 3-10.

[0011] Furthermore, the concentration of acetaldehyde added to the buffer solution is 0.01-0.1 mM.

[0012] Furthermore, the mass volume ratio of the enzyme-containing catalyst to the buffer solution is 0.5-5 mg / mL.

[0013] The present invention synthesizes L-lactic acid with CO2 as raw material by coupling the synthesis of a synthetic enzyme-containing catalyst with the regeneration of a cofactor (NADH), and can obtain a high catalytic effect under very mild conditions, and has a good market prospect; in the enzymatic reaction cycle, pyruvate decarboxylase hydrogenates CO2 and acetaldehyde to generate pyruvate. Under light conditions, Co-TCPP in the enzyme-containing catalyst dissociates H2O into H + and O 2- , oxidized coenzyme I (NAD + ) + Reduction generates NADH, which acts as a hydrogen donor. Under the action of L-lactate dehydrogenase, pyruvate is reduced to L-lactic acid, which is then oxidized to generate NAD + Compared with other NADH regeneration technologies, it saves the use of ADH enzyme and further reduces costs.

[0014] The present invention uses a synthesized enzyme-containing catalyst to compound Co-TCPP, and on the basis of a large-pore MOF loaded with enzyme, enhances its photocatalytic activity and can realize the dual-function NADH and NAD + The photoregeneration of Figure 1 .

[0015] The present invention adopts light-enzyme coupling technology, so that carbon dioxide can be converted into high value-added organic L-lactic acid at room temperature, i.e., 25°C. On the one hand, embedding the enzyme in the MOF structure not only reduces the reaction cost by recycling, but also causes little damage to the enzyme, and is conducive to the correct conformational folding of the enzyme protein and the unfolding of the active center. In addition, the porous ZIF-8 can provide more substrate channels and enzyme contact sites, which is very beneficial to the catalytic reaction. On the other hand, the direct integration of the photogenerated electron system with the LDH enzyme that consumes NADH shortens the transmission distance between the substrate and the electron, the substrate and the hole, and the electron and the electron, which is also helpful to improve the reaction efficiency, and improves the reaction efficiency. Compared with the free system, the efficiency is increased by 2.03 times. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 It is a flow chart of the reaction process of the present invention; Figure 2 is the X-ray diffraction pattern of the catalyst synthesized in Example 1 of the present invention; Figure 3 1 is a high-resolution transmission electron micrograph of the catalyst synthesized in Example 1 of the present invention; (a) is a STEM image; (b)-(f) are element distribution diagrams. DETAILED DESCRIPTION

[0017] The following are specific embodiments of the present invention and the accompanying drawings to further describe the technical solution of the present invention, but the present invention is not limited to these embodiments.

[0018] Example 1 Take 4.1 g of 2-methylimidazole and dissolve it in 40 mL of deionized water, then add 3% Co-TCPP of MOF mass, add 3 mL of L-lactate dehydrogenase (LDH) and pyruvate decarboxylase (PyDc) (enzyme liquid is 2 mg / mL), and after fully dissolved, keep the solution at a constant temperature and speed device with a stirring speed of 30 °C and 400 rpm. Then, add 1 mL of zinc nitrate hexahydrate (0.31 mol / L) and 16 μL of PVP (1 mg / mL) dropwise. Stir, wash and dry. The enzyme-containing catalyst PyDc&LDH&Co-TCPP@ZIF-8 was prepared, recorded as 2E&Co-TCPP@ZIF-8.

[0019] like Figure 2 , Figure 3 As shown in the XRD pattern and HRTEM pattern of the catalyst synthesized in Example 1, it can be seen that the catalyst prepared in Example 1 presents regular particles and the doped Co element is highly dispersed.

[0020] Example 2 Co-TCPP was added at 0.1% by weight of MOF, and the rest was the same as in Example 1 to prepare PyDc&LDH&Co-TCPP@ZIF-8 catalyst, which was recorded as 2E&Co-TCPP@ZIF-8*1.

[0021] Example 3 Co-TCPP was added at 1% by weight of MOF, and the rest was the same as in Example 1 to prepare PyDc&LDH&Co-TCPP@ZIF-8 catalyst, recorded as 2E&Co-TCPP@ZIF-8*2.

[0022] Example 4 Co-TCPP was added at 5% by weight of MOF, and the rest was the same as in Example 1 to prepare PyDc&LDH&Co-TCPP@ZIF-8 catalyst, recorded as 2E&Co-TCPP@ZIF-8*3.

[0023] Example 5 like Figure 1As shown, a method for converting CO2 as a raw material into L-lactic acid comprises the following steps: (1) Mix acetaldehyde and phosphate buffer in proportion, and inject CO2 into the mixture until saturated; (2) adding the enzyme-containing catalyst prepared in Example 1 to the mixed solution of step (1); (3) Add oxidized coenzyme NAD to the mixture in step (2) + ; Simulated illumination under xenon lamp conditions, wavelength λ ≥ 420 nm; The amount of phosphate buffer added was 30 mL, the amount of catalyst used was 1.5 mg / ml, the amount of acetaldehyde added was 0.2 mM, and the amount of NAD + The added amount was 1 mM, and the buffer pH was 8; the reaction product was obtained and numbered A1.

[0024] During the reaction, the concentration of L-lactic acid in the reaction solution was detected every 2 hours.

[0025] Example 6 The catalyst of Example 1 added in Example 5 was replaced by the catalyst of Examples 2-4, and other conditions remained unchanged to obtain reaction products numbered A2-A4.

[0026] Example 7 The pH of the buffer solution in Example 5 was changed to 5.8, 6.4, 7, 7.6, and 9 to obtain reaction products numbered A5-A9.

[0027] Comparative Example 1 In the process of preparing the catalyst in Example 1, L-lactate dehydrogenase (LDH) and pyruvate decarboxylase (PyDc) were not added to obtain the Co-TCPP@ZIF-8 catalyst.

[0028] A method for converting CO2 as a raw material into L-lactic acid comprises the following steps: (1) Mix acetaldehyde and phosphate buffer in proportion, and inject CO2 into the mixture until saturated; (2) Adding Co-TCPP@ZIF-8 to the mixed solution in step (1); (3) Add oxidized coenzyme NAD to the mixture in step (2) + ; (4) Add 1-5 mL of L-lactate dehydrogenase (LDH) and pyruvate decarboxylase (PyDc) (enzyme liquid is 1-5 mg / mL); Simulated illumination under xenon lamp conditions, wavelength λ ≥ 420 nm; The amount of phosphate buffer added was 30 mL, the amount of catalyst used was 1.5 mg / ml, the amount of acetaldehyde added was 0.2 mM, and the amount of NAD + The added amount was 1 mM, and the buffer pH was 8; the reaction product was obtained and numbered A10.

[0029] During the reaction, the concentration of L-lactic acid in the reaction solution was detected every 2 hours.

[0030] The evaluation results of the CO2 synthesis of L-lactic acid reaction in Examples 1-7 are shown in Table 1.

[0031] Table 1

[0032] It can be seen from Table 1 that when the Co-TCPP loading is 3% and the pH is 8, the reaction is performed for 8 hours, so that the L-lactic acid concentration is the largest, which is 40.2 μM. The L-lactic acid concentration of Comparative Example 1 is the lowest, and the L-lactic acid concentration of the embodiment is twice that of Comparative Example 1.

[0033] For matters not mentioned above, the prior art applies.

[0034] Although some specific embodiments of the present invention have been described in detail through examples, those skilled in the art should understand that the above examples are for illustration only and are not intended to limit the scope of the present invention. Those skilled in the art to which the present invention belongs may make various modifications or supplements to the specific embodiments described or replace them in a similar manner, but they will not deviate from the direction of the present invention or exceed the scope defined by the attached claims. Those skilled in the art should understand that any modifications, equivalent substitutions, improvements, etc. made to the above embodiments based on the technical essence of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing an enzyme-containing catalyst, characterized in that: Co-TCPP, L-lactate dehydrogenase and pyruvate decarboxylase were added to the 2-methylimidazole aqueous solution. After they were fully dissolved, zinc nitrate hexahydrate solution and polyvinyl pyrrolidone solution were added dropwise. The mixture was stirred to produce a precipitate, which was washed and dried to obtain the enzyme-containing catalyst PyDc&LDH&Co-TCPP@ZIF-8.

2. The preparation method according to claim 1, characterized in that: The mass of Co-TCPP is 0.1%-5% of the mass of the generated ZIF-8.

3. The preparation method according to claim 1, characterized in that: The molar mass ratio of zinc nitrate hexahydrate to L-lactate dehydrogenase and pyruvate decarboxylase is 0.2-0.35 mmol: 5-7 mg: 5-7 mg.

4. The preparation method according to claim 1, characterized in that: The added L-lactate dehydrogenase and pyruvate decarboxylase are enzyme solutions, and the concentrations of the two enzyme solutions are 1-5 mg / mL.

5. An enzyme-containing catalyst prepared by the preparation method according to any one of claims 1 to 4.

6. Use of the enzyme-containing catalyst as claimed in claim 5 in the preparation of L-lactic acid, characterized in that: CO2 was added to the buffer until saturated, and acetaldehyde, enzyme catalyst and oxidized coenzyme NAD were added. + , provide light conditions, and convert CO2 as raw material into L-lactic acid.

7. The use according to claim 6, characterized in that: The buffer is a phosphate buffer with a pH of 3-10.

8. The use according to claim 6, characterized in that: The concentration of acetaldehyde added to the buffer was 0.01-0.1 mM.

9. The use according to claim 6, characterized in that: The mass volume ratio of the enzyme-containing catalyst to the buffer solution is 0.5-5 mg / mL.

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