A multi-enzyme cascade method for catalyzing synthesis of urea from co2 and nh3 and application thereof

Abstract

The present application relates to a kind of catalytic CO2 and NH3 synthesis urea multi-enzyme cascade method and its application, and CO2 and NH3 are synthesized carbamic acid, it is converted into carbamoyl phosphate by carbamoyl phosphate kinase, carbamoyl phosphate is generated citrulline with ornithine by ornithine carbamoyltransferase catalysis, citrulline is converted into arginine with NH3 by arginine synthetase, and arginine is generated urea and ornithine by arginase catalysis.The catalytic reaction method of the present application is simple, and effectively shorten the reaction step of CO2 and NH3 synthesis urea, realize the high-efficiency conversion of CO2 and NH3 under mild conditions Synthesis urea, provide new green technology route for the full-chain biosynthesis urea with CO2 and NH3 as raw material.

Description

Technical Field

[0001] This invention relates to the field of biocatalysis technology, and in particular to a multi-enzyme cascade method for catalyzing the synthesis of urea from CO2 and NH3, and its application. Background Technology

[0002] Urea is one of the simplest organic compounds and a major nitrogenous end product of protein metabolism in mammals and certain fish. As an important nitrogenous compound, urea is the most widely used nitrogen fertilizer in the world, suitable for various soils and plants. It is easy to store and use, has minimal impact on soil, and is one of the most widely used chemical nitrogen fertilizers, as well as the one with the highest nitrogen content. Furthermore, urea is also a widely used industrial raw material, used extensively in the production of melamine, urea-formaldehyde resin, monosodium glutamate, and many other products, demonstrating its extremely broad applications.

[0003] Industrial urea production primarily involves the direct synthesis of urea from carbon dioxide and ammonia under high temperature and pressure. This process suffers from complex preparation methods, high energy consumption, and high carbon emissions. In recent years, electro / photocatalytic synthesis of urea from carbon dioxide and nitrogen has emerged as a green alternative. It offers milder reaction conditions and directly utilizes two low-cost, widely available gases, carbon dioxide and nitrogen, aligning with the "dual carbon" goal. However, due to the inert nature of both carbon dioxide and nitrogen, activation is challenging, and current photo / electrocatalytic urea synthesis technologies still face limitations in catalytic activity and urea selectivity. Enzymatic catalysis, on the other hand, can efficiently activate inert molecules like carbon dioxide under mild conditions and achieve CN-C bond coupling, with a single reaction product, thus demonstrating greater potential and advantages in terms of economy and sustainability.

[0004] To date, the only reported natural metabolic pathway involving urea synthesis in organisms is the urea (ornithine) cycle. This pathway utilizes a cascade of carbamoyl phosphate synthase, ornithine carbamoyltransferase, argininosuccinate synthase, argininosuccinate lyase, and argininase to catalyze the metabolism of carbon dioxide and ammonia to produce urea. Therefore, based on the natural metabolic pathway of the urea cycle, a novel and efficient artificial urea synthesis pathway should be designed and constructed using synthetic biology. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides a multi-enzyme cascade method for catalyzing the synthesis of urea from CO2 and NH3, and its application. Compared to the urea cycle, this pathway first reduces the consumption of cofactors ATP and glutamine through carbamoyl phosphokinase, and then catalyzes the reaction of citrulline and NH3 to generate the intermediate product arginine in one step through arginine synthase. This successfully shortens the effective reaction steps for the synthesis of urea from CO2 and NH3, achieving efficient conversion of CO2 and NH3 into urea under mild conditions. This provides a novel green technical route for the full-chain biosynthesis of urea using CO2 and NH3 as raw materials.

[0006] To achieve this objective, the present invention adopts the following technical solution: In a first aspect, the present invention provides a multi-enzyme cascade method for catalyzing the synthesis of urea from CO2 and NH3, the method comprising: synthesizing carbamic acid from CO2 and NH3, converting it to carbamic acid via carbamoyl phosphokinase, generating citrulline from carbamoyl phosphokinase and ornithine via ornithine carbamoyltransferase, converting citrulline and NH3 into arginine via arginine synthase, and generating urea and ornithine from arginine via arginase.

[0007] In this invention, CO2 and NH3 are used as raw materials. First, CO2 and NH3 are converted into the active substance carbamoyl phosphate by carbamoyl phosphate kinase. Carbamoyl phosphate and ornithine are then catalyzed by ornithine carbamoyltransferase to generate citrulline. Citrulline and NH3 are then converted into the intermediate product arginine by arginine synthase. Finally, arginine is hydrolyzed by arginase to generate urea and ornithine. The obtained ornithine can be used as an initiator to synthesize citrulline with carbamoyl phosphate and continue to enter the same pathway to achieve the recycling of urea and ornithine.

[0008] Preferably, the carbamoyl phosphokinase comprises a derivative of TbCK (Thermophilic Coccus). Thermococcus barophilus ) 、 PfCK (Fireball Bacterium) Pyrococcus furiosus ), TsCK (Siberian Thermococcus) Thermococcus sibiricus ) or FnCK (Bacillus multiscintillans) Fervidobacterium nodosum Any one or at least two of the following.

[0009] In a specific embodiment of the present invention, the carbamoyl phosphokinase used is TbCK. In the present invention, enzymes with the function of catalyzing the conversion of carbamate or carbamate into carbamoyl phosphokinase can be used in the above-mentioned pathway.

[0010] Preferably, the ornithine carbamoyltransferase comprises TsOTC (derived from Thermococcus siberianis). Thermococcus sibiricu s).

[0011] In a specific embodiment of the present invention, the ornithine carbamoyltransferase used is TsOTC. In the present invention, ornithine carbamoyltransferase is any enzyme that can catalyze the conversion of carbamoyl phosphate and ornithine into citrulline and can be used in the above-mentioned pathway.

[0012] Preferably, the arginine synthase comprises TpArcE (Thermococcus proteoglycanus). Thermococcus peptonophilus ), AbArcE (anaerobic filamentous bacteria) Anaerolineales bacterium CzArcE (Rose-modified bacillus) Melioribacter roseus MrArcE Candidatus Zixiibacteriota ).

[0013] In a specific embodiment of the present invention, the arginine synthase used is TpArcE. In the present invention, any enzyme that can catalyze the conversion of citrulline and NH3 into arginine can be used in the above-mentioned pathway.

[0014] Preferably, the arginase includes BsARS (Bacillus subtilis). Bacillus subtilis ).

[0015] In a specific embodiment of the present invention, the arginase used is BsARS. In the present invention, any enzyme that can catalyze the hydrolysis of arginine into urea and ornithine can be used in the above-mentioned pathway.

[0016] In a second aspect, the present invention provides an application of the multi-enzyme cascade method for catalyzing CO2 and NH3 according to the first aspect in the synthesis of urea.

[0017] Thirdly, the present invention provides a multi-enzyme cascade method for catalyzing the synthesis of urea from CO2 and NH3, the method comprising synthesizing urea using the multi-enzyme cascade reaction pathway for catalyzing the synthesis of urea from CO2 and NH3 as described in the first aspect.

[0018] In one specific embodiment of the present invention, carbamoyl phosphokinase is used to convert carbamoic acid, which is spontaneously formed from CO2 and NH3 in water, into the active substance carbamoyl phosphate through a phosphate transfer reaction; then the generated carbamoyl phosphate undergoes a transamination reaction with ornithine to convert into citrulline; further, citrulline reacts with NH3 under the catalysis of arginine synthase to generate arginine; finally, arginine is hydrolyzed by arginase to obtain urea and ornithine.

[0019] Preferably, the method includes mixing substrate 1, substrate 2, complex enzyme, ornithine, ATP, MgCl2 and MnCl2 in a buffer solution for reaction.

[0020] Preferably, the substrate 1 includes CO2 and / or NaHCO3.

[0021] Preferably, the substrate 2 includes any one or a combination of at least two of NH3, NH3·H2O and NH4Cl.

[0022] Preferably, the complex enzyme comprises carbamoyl phosphokinase, ornithine carbamoyltransferase, arginine synthase, and arginase.

[0023] Preferably, the mass ratio of carbamoyl phosphokinase, ornithine carbamoyltransferase, arginine synthase, and arginase is (0.2-1):0.2:(0.2-1):(0.2-1). The (0.2-1) can be, for example, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.

[0024] Preferably, the concentration of the complex enzyme is 0.8-1.2 mg / mL. The 0.8-1.2 mg / mL can be, for example, 0.8 mg / mL, 0.9 mg / mL, 1.0 mg / mL, 1.1 mg / mL, or 1.2 mg / mL.

[0025] Preferably, the concentration of ornithine is 0.5-20 mM. The 0.5-20 mM can be, for example, 0.5 mM, 1 mM, 2 mM, 4 mM, 6 mM, 8 mM, 10 mM, 12 mM, 14 mM, 16 mM, 18 mM or 20 mM, etc.

[0026] Preferably, the concentration of ATP is 1-100 mM. The 1-100 mM can be, for example, 1 mM, 5 mM, 10 mM, 20 mM, 40 mM, 60 mM, 80 mM or 100 mM.

[0027] Preferably, the concentration of MgCl2 is 0.5-10 mM. The 0.5-10 mM can be, for example, 0.5 mM, 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM or 10 mM, etc.

[0028] Preferably, the concentration of MnCl2 is 0.1-5 mM. The 0.1-5 mM can be, for example, 0.1 mM, 1 mM, 2 mM, 3 mM, 4 mM, or 5 mM.

[0029] Preferably, when substrate 1 is NaHCO3, the concentration of substrate 1 is 5-200 mM. The 5-200 mM can be, for example, 5 mM, 10 mM, 50 mM, 100 mM, 150 mM, or 200 mM.

[0030] Preferably, when the substrate 1 is CO2, the flow rate of the substrate 1 is 5-15 mL / min. The 5-15 mL / min can be, for example, 5 mL / min, 7 mL / min, 10 mL / min, 12 mL / min, or 15 mL / min.

[0031] Preferably, when substrate 2 is NH3·H2O or NH4Cl, the concentration of substrate 2 is 5-100 mM. The 5-100 mM can be, for example, 5 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, or 100 mM.

[0032] Preferably, when the substrate 2 is NH3, the amount of substrate 2 added is 10-100 mM. The 10-100 mM can be, for example, 10 mM, 20 mM, 40 mM, 60 mM, 80 mM, or 100 mM.

[0033] Preferably, the mixing reaction time is 1-6 hours. The 1-6 hours can be, for example, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, or 6 hours.

[0034] Preferably, the buffer solution includes any one or a combination of at least two of PB buffer, Tris-HCl buffer, HEPES buffer, or borate buffer.

[0035] Preferably, the concentration of the buffer solution is 20-100 mM. The 20-100 mM can be, for example, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, or 100 mM.

[0036] Preferably, the reaction temperature is 35-45 °C and the time is 1-24 h. The 35-45 °C can be, for example, 35 °C, 37 °C, 39 °C, 41 °C, 43 °C, or 45 °C. The 1-24 h can be, for example, 1 h, 2 h, 3 h, 4 h, 5 h, 6 h, 7 h, 8 h, 9 h, 10 h, 11 h, 12 h, 13 h, 14 h, 15 h, 16 h, 17 h, 18 h, 19 h, 20 h, 21 h, 22 h, 23 h, or 24 h.

[0037] Compared with the prior art, the present invention has at least the following beneficial effects: Unlike the natural urea synthesis pathway, the multi-enzyme cascade catalytic synthesis of urea from CO2 and NH3 based on arginine synthase in a one-step catalysis of citrulline and NH3 to produce the intermediate product arginine is the shortest cell-free artificial urea synthesis pathway reported to date. It can achieve efficient conversion of CO2 and NH3 to urea under mild conditions, laying a technical foundation for CO2 and NH3 as carbon / nitrogen carriers to drive the transformative development of important fields of biochemical engineering. Attached Figure Description

[0038] Figure 1 This diagram illustrates the pathway for the synthesis of urea from CO2 and NH3 via a multi-enzyme cascade reaction.

[0039] Figure 2 This is a flowchart illustrating the reaction process of a multi-enzyme cascade reaction catalyzing the synthesis of the intermediate product citrulline from sodium bicarbonate and ammonium chloride.

[0040] Figure 3 This is a flowchart illustrating the reaction process of a multi-enzyme cascade catalyzing the synthesis of arginine, an intermediate product, from sodium bicarbonate and ammonium chloride.

[0041] Figure 4 This is a diagram illustrating the reaction process of sodium bicarbonate and ammonium chloride synthesizing urea via a multi-enzyme cascade reaction.

[0042] Figure 5 The graph shows the relative yields of urea synthesized by multi-enzyme cascade under different carbamoyl phosphokinase concentrations. Detailed Implementation

[0043] The technical solution of the present invention will be further described below with reference to the accompanying drawings and specific embodiments. However, the following examples are merely simplified examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention is determined by the claims.

[0044] Example 1 This embodiment constructs a pathway for the synthesis of urea from CO2 and NH3 via a multi-enzyme cascade reaction. This invention is based on the one-step catalytic reaction of citrulline and NH3 to produce the intermediate product arginine using arginine synthase. The constructed multi-enzyme cascade reaction pathway for the synthesis of urea from CO2 and NH3 includes: using CO2 and NH3 as raw materials, CO2 and NH3 are converted into the active substance carbamoyl phosphate by carbamoyl phosphokinase; then, carbamoyl phosphate and ornithine are converted into citrulline under the catalysis of ornithine carbamoyltransferase; further, citrulline and NH3 are converted into the intermediate product arginine by arginine synthase; finally, arginine is hydrolyzed by arginase to produce urea and ornithine, realizing the recycling of urea and ornithine. The specific process is as follows: Figure 1 As shown.

[0045] Example 2 This embodiment verifies the urea synthesis pathway. (1) Synthesis of citrulline At 40℃, 100 mM substrate NaHCO3, 100 mM NH4Cl, 0.2 mg / mL carbamoyl phosphokinase TbCK, 0.3 mg / mL ornithine carbamoyltransferase TsOTC, 20 mM ATP, 1.0 mM ornithine, and 5 mM MgCl2 were added to a total system volume of 500 μL Tris-HCl buffer (50 mM, pH 8.5). The reaction was stopped after 6 h. The concentration of citrulline was determined by pre-column derivatization and high performance liquid chromatography with a fluorescence detector (Shimadzu HPLC LC20A). The total volume of the derivatization system was 1 mL, containing 600 μL borate buffer, 200 μL methanol, 130 μL H2O, 60 μL sample, and 10 μL diethyl ethoxymethylene malonate (DEEMM). The derivatization reaction was incubated at room temperature for 10 min, followed by chromatography using a C18 column (Shim-pack GIST-HP-C18, 2.1 mm × 100 mm × 3 μm) and a fluorescence detector λ. ex =1.41 mM citrulline was detected at a column density of 284 nm and a column temperature of 35 °C. The reaction process for citrulline formation concentration is as follows. Figure 2 As shown.

[0046] (2) Synthesis of arginine At 40℃, 100 mM substrate NaHCO3, 100 mM NH4Cl, 0.2 mg / mL carbamoyl phosphokinase TbCK, 0.3 mg / mL ornithine carbamoyltransferase TsOTC, 0.3 mg / mL arginine synthase TpArcE, 20 mM ATP, 1.0 mM ornithine, and 5 mM MgCl2 were added to a total system volume of 500 μL Tris-HCl buffer (50 mM, pH 8.5). The reaction was stopped after 6 h. The arginine concentration was determined by pre-column derivatization and high performance liquid chromatography with a fluorescence detector (Shimadzu HPLC LC20A). The total volume of the derivatization system was 1 mL, containing 600 μL borate buffer, 200 μL methanol, 130 μL H2O, 60 μL sample, and 10 μL diethyl ethoxymethylene malonate (DEEMM). The derivatization reaction was incubated at room temperature for 10 min, followed by chromatography using a C18 column (Shim-pack GIST-HP-C18, 2.1 mm × 100 mm × 3 μm) and a fluorescence detector λ. ex Arginine was detected at 4.48 mM under the conditions of 284 nm and column temperature of 35 ℃. The reaction process for arginine formation concentration is as follows. Figure 3 As shown.

[0047] (3) Synthetic urea At 40℃, 100 mM substrate NaHCO3, 100 mM NH4Cl, 0.2 mg / mL carbamoyl phosphokinase TbCK, 0.3 mg / mL ornithine carbamoyltransferase TsOTC, 0.3 mg / mL arginine synthase TpArcE, 0.1 mg / mL arginase BsARS, 20 mM ATP, 1.0 mM ornithine, 5 mM MgCl2, and 2.0 mM MnCl2 were added to a 500 μL Tris-HCl buffer (50 mM, pH 8.5). The reaction was stopped after 6 h. The urea concentration was determined using the pre-column derivatization reagent 9-hydroxyton and high-performance liquid chromatography with a fluorescence detector (Shimadzu HPLC LC20A). The derivatizing reagent was prepared by dissolving 198 mg of 9-hydroxytoluene powder in 50 mL of n-propanol. The total volume of the derivatization system was 1 mL, containing 100 μL of hydrochloric acid (1.5 M), 400 μL of sample, and 0.2 M of 9-hydroxytoluene. After shaking well, derivatization was performed at room temperature in the dark for 30 min. Then, a C18 column (Shim-pack GIST-HP-C18, 2.1 mm × 100 mm × 3 μm) was used with a fluorescence detector λ. ex =3.20 mM urea was detected at a column density of 213 nm and a column temperature of 35 °C. The reaction process for urea formation concentration is as follows. Figure 4 As shown.

[0048] Example 3 This embodiment investigates the effect of different concentrations of carbamoyl phosphokinase on the synthesis of urea. At 40℃, in a total volume of 500 μL Tris-HCl buffer (50 mM, pH 8.5), 100 mM substrate NaHCO3, 100 mM NH4Cl, different concentrations (0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL) of carbamoyl phosphokinase TbCK, 0.3 mg / mL ornithine carbamoyltransferase TsOTC, 0.3 mg / mL arginine synthase TpArcE, 0.1 mg / mL arginase BsARS, 20 mM ATP, 1.0 mM ornithine, 5 mM MgCl2, and 2.0 mM MnCl2 were added. The reaction was stopped after 6 h. The urea concentration was determined using the pre-column derivatization reagent 9-hydroxyton and high performance liquid chromatography with a fluorescence detector (Shimadzu HPLC LC20A). The derivatizing reagent was prepared by dissolving 198 mg of 9-hydroxytoluene powder in 50 mL of n-propanol. The total volume of the derivatization system was 1 mL, containing 100 μL of hydrochloric acid (1.5 M), 400 μL of sample, and 0.2 M of 9-hydroxytoluene. After shaking well, derivatization was performed at room temperature in the dark for 30 min. Then, a C18 column (Shim-pack GIST-HP-C18, 2.1 mm × 100 mm × 3 μm) was used with a fluorescence detector λ. ex The highest urea concentration was detected at a column density of 213 nm and a column temperature of 35 °C, with a concentration of 0.4 mg / mL carbamoyl phosphokinase. Figure 5 As shown.

[0049] Example 4 This embodiment uses CO2 and NH3 as substrates to synthesize urea. At 30℃, the Tris-HCl buffer (50 mM, pH 8.5) was first degassed and saturated with CO2 (gas flow rate 10 mL / min) for 1 h. Then, 100 mM substrate NH3·H2O, 0.2 mg / mL carbamoyl phosphokinase TbCK, 0.3 mg / mL ornithine carbamoyltransferase TsOTC, 0.3 mg / mL arginine synthase TpArcE, 0.1 mg / mL arginase BsARS, 20 mM ATP, 1.0 mM ornithine, 5 mM MgCl2, and 2.0 mM MnCl2 were added to a total volume of 500 μL of Tris-HCl buffer. The reaction was continued to be aerated for 6 h to terminate the reaction. The urea concentration was determined using the pre-column derivatization reagent 9-hydroxyton and measured by high performance liquid chromatography with a fluorescence detector (Shimadzu HPLC LC20A). The derivatizing reagent was prepared by dissolving 198 mg of 9-hydroxytoluene powder in 50 mL of n-propanol. The total volume of the derivatization system was 1 mL, containing 100 μL of hydrochloric acid (1.5 M), 400 μL of sample, and 0.2 M of 9-hydroxytoluene. After shaking well, derivatization was performed at room temperature in the dark for 30 min. Then, a C18 column (Shim-pack GIST-HP-C18, 2.1 mm × 100 mm × 3 μm) was used with a fluorescence detector λ. ex =213 nm, column temperature 35 o 0.3 mM urea was detected under C conditions.

[0050] Example 5 This embodiment investigates the effects of different types of arginine synthase on urea precursor production. At 40℃, 100 mM substrate NH4HCO3, 0.2 mg / mL carbamoyl phosphokinase TbCK, 0.3 mg / mL ornithine carbamoyltransferase TsOTC, 0.3 mg / mL arginine synthase (any one of TpArcE, AbArcE, CzArcE, or MrArcE), 20 mM ATP, 1.0 mM ornithine, and 5 mM MgCl2 were added to a total volume of 500 μL Tris-HCl buffer. The reaction was stopped after 1 h. The arginine concentration was determined by pre-column derivatization and high performance liquid chromatography with a fluorescence detector (Shimadzu HPLC LC20A). The total volume of the derivatization system was 1 mL, containing 600 μL borate buffer, 200 μL methanol, 130 μL H2O, 60 μL sample, and 10 μL diethyl ethoxymethylene malonate (DEEMM). The derivatization reaction was incubated at room temperature for 10 min, followed by chromatography using a C18 column (Shim-pack GIST-HP-C18, 2.1 mm × 100 mm × 3 μm) and a fluorescence detector λ. exThe arginine concentrations detected at 284 nm and a column temperature of 35 °C were TpArcE: 1.1 mM; AbArcE: 3.4 mM; CzArcE: 2.9 mM; and MrArcE: 2.3 mM. These results indicate that using these four enzymes can achieve good results.

[0051] In summary, the multi-enzyme cascade method for catalyzing the synthesis of urea from CO2 and NH3 constructed in this invention utilizes arginine synthase to catalyze the reaction of citrulline and NH3 to generate the intermediate product arginine in one step, successfully shortening the reaction steps for the synthesis of urea from CO2 and NH3, and can efficiently catalyze the conversion of CO2 and NH3 into urea.

[0052] The applicant declares that the above description is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention fall within the protection and disclosure scope of the present invention.

Claims

1. A multi-enzyme cascade reaction method for catalyzing the synthesis of urea from CO2 and NH3, characterized in that, The method includes: synthesizing carbamic acid from CO2 and NH3, converting it to carbamic phosphate via carbamoyl phosphokinase, generating citrulline from carbamoyl phosphate and ornithine via ornithine carbamoyltransferase, converting citrulline and NH3 into arginine via arginine synthase, and generating urea and ornithine from arginine via arginase.

2. The multi-enzyme cascade method for catalytic synthesis of urea from CO2 and NH3 according to claim 1, characterized in that, The carbamoyl phosphokinase includes any one or a combination of at least two of TbCK, PfCK, TsCK, or FnCK.

3. The multi-enzyme cascade method for catalytic synthesis of urea from CO2 and NH3 according to claim 1 or 2, characterized in that, The ornithine carbamoyltransferase includes TsOTC; Preferably, the arginine synthase comprises any one or a combination of at least two of TpArcE, AbArcE, CzArcE, or MrArcE; Preferably, the arginase comprises BsARS.

4. The application of a multi-enzyme cascade method for catalytic CO2 and NH3 according to any one of claims 1-3 in the synthesis of urea.

5. A multi-enzyme cascade method for catalyzing the synthesis of urea from CO2 and NH3, characterized in that, The method includes synthesizing urea using the multi-enzyme cascade reaction pathway for the catalytic synthesis of urea from CO2 and NH3 as described in any one of claims 1-3.

6. The multi-enzyme cascade method for catalytic synthesis of urea from CO2 and NH3 according to claim 5, characterized in that, The method involves mixing substrate 1, substrate 2, complex enzyme, ornithine, ATP, MgCl2 and MnCl2 in a buffer solution and reacting them.

7. The multi-enzyme cascade method for catalytic synthesis of urea from CO2 and NH3 according to claim 5, characterized in that, The substrate 1 includes CO2 and / or NaHCO3; Preferably, the substrate 2 includes any one or a combination of at least two of NH3, NH3·H2O and NH4Cl.

8. The multi-enzyme cascade method for catalytic synthesis of urea from CO2 and NH3 according to claim 6 or 7, characterized in that, The complex enzyme includes carbamoyl phosphokinase, ornithine carbamoyltransferase, arginine synthase, and arginase. Preferably, the mass ratio of carbamoyl phosphokinase, ornithine carbamoyltransferase, arginine synthase and arginase is (0.2-1):0.2:(0.2-1):(0.2-1).

9. The multi-enzyme cascade method for catalytic synthesis of urea from CO2 and NH3 according to any one of claims 6-8, characterized in that, The concentration of the complex enzyme is 0.8-1.2 mg / mL; Preferably, the concentration of ornithine is 0.5-20 mM; Preferably, the concentration of ATP is 1-100 mM; Preferably, the concentration of MgCl2 is 0.5-10 mM; Preferably, the concentration of MnCl2 is 0.1-5 mM; Preferably, when substrate 1 is NaHCO3, the concentration of substrate 1 is 5-200 mM; Preferably, when substrate 1 is CO2, the flow rate of substrate 1 is 5-15 mL / min; Preferably, when substrate 2 is NH3·H2O or NH4Cl, the concentration of substrate 2 is 5-100 mM; Preferably, when the substrate 2 is NH3, the amount of substrate 2 added is 10-100 mM; Preferably, the mixing reaction takes 1-6 hours.

10. The multi-enzyme cascade method for catalytic synthesis of urea from CO2 and NH3 according to any one of claims 6-9, characterized in that, The buffer solution includes any one or a combination of at least two of PB buffer, Tris-HCl buffer, HEPES buffer, or borate buffer. Preferably, the concentration of the buffer solution is 20-100 mM; Preferably, the reaction temperature is 35-45 °C and the time is 1-24 h.

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