Method for synthesizing glucose through photo-enzyme cascade catalysis

Abstract

The invention relates to the technical field of glucose synthesis, and provides a method for synthesizing glucose through photo-enzyme cascade catalysis, which comprises the following steps: carrying out photo-catalytic reaction on oxidized coenzyme to obtain reduced coenzyme; taking a carbon dioxide source and a pentose compound as raw materials, performing a first series of enzyme catalytic reactions, and taking the reduced coenzyme as a substrate in the process of the first series of enzyme catalytic reactions to obtain glyceraldehyde-3-phosphoric acid and ketose dihydroxyacetone phosphate; and by taking the glyceraldehyde-3-phosphoric acid and the ketose dihydroxyacetone phosphoric acid as raw materials, carrying out a second series of enzyme catalytic reactions to obtain the glucose. According to the method for synthesizing glucose through photo-enzyme cascade catalysis, two green processes of photo-catalysis and enzyme catalysis are combined, the production period is greatly shortened, the product selectivity and yield are improved, and a new way is opened up for the green synthesis technology of glucose.

Description

Technical Field

[0001] The present application belongs to the technical field of glucose synthesis, and in particular to a method for synthesizing glucose by photoenzyme cascade catalysis. Background Art

[0002] Glucose is an important carbohydrate that is widely used in the food industry, the pharmaceutical field, and biofuel production. There are two main ways to produce glucose: one is through starch hydrolysis, and the other is through bioconversion from plants and most algae. The former requires high energy consumption conditions of high temperature and high pressure, and also produces wastewater and waste gas, causing environmental problems; the latter is through photosynthesis, has a long production cycle, low yield, and is easily restricted by climate and region. Summary of the invention

[0003] The purpose of the present application is to provide a method for synthesizing glucose by photoenzyme cascade catalysis, aiming to solve the problems of high energy consumption, long production cycle and low yield of existing glucose synthesis methods.

[0004] In order to achieve the above application purpose, the technical solution adopted in this application is as follows:

[0005] The present application provides a method for synthesizing glucose by photoenzyme cascade catalysis, comprising the following steps:

[0006] The oxidized coenzyme is subjected to a photocatalytic reaction to obtain a reduced coenzyme;

[0007] A first series of enzyme catalytic reactions are carried out using a carbon dioxide source and a pentose compound as raw materials, and the reduced coenzyme is used as a substrate during the first series of enzyme catalytic reactions to obtain glyceraldehyde 3-phosphate and ketose dihydroxyacetone phosphate;

[0008] The glyceraldehyde 3-phosphate and the ketose dihydroxyacetone phosphate are used as raw materials to carry out a second series of enzyme catalytic reactions to obtain glucose.

[0009] The present application provides a method for synthesizing glucose by photoenzyme cascade catalysis, which utilizes photocatalytic technology to generate reduced coenzymes, and the generated reduced coenzymes are combined with other enzymes to carry out a series of enzyme-catalyzed reactions, thereby realizing the photoenzyme coupling catalysis to synthesize glucose. Since each enzyme is selective for the substrates and products of its reaction, it ensures that a series of enzyme-catalyzed reaction processes are highly regulated, thereby realizing the efficient and directional selective conversion of carbon dioxide to glucose. The present application combines the two green process technologies of photocatalysis and enzyme catalysis. The reaction rates of photocatalysis and enzyme catalysis are both at the millisecond level, and artificial synthesis is non-cellular synthesis, which does not require the cultivation of organisms, greatly shortening the production cycle. The electronic intermediates and enzymes in the reaction have structural specificity and can provide specific sites, greatly improving product selectivity, and opening up new avenues for the green synthesis technology of glucose. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0011] Figure 1 It is a reaction route diagram of a method for synthesizing glucose by photoenzyme cascade catalysis provided in an embodiment of the present application;

[0012] Figure 2 This is the UV-visible absorption spectrum of the solution before and after the photocatalytic reaction provided in Example 1 of the present application. DETAILED DESCRIPTION

[0013] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present application more clearly understood, the present application is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and are not used to limit the present application.

[0014] In this application, the term "and / or" describes the association relationship of associated objects, indicating that there may be three relationships. For example, A and / or B can mean: A exists alone, A and B exist at the same time, and B exists alone. A and B can be singular or plural. The character " / " generally indicates that the associated objects are in an "or" relationship.

[0015] In this application, "at least one" means one or more, and "plurality" means two or more. "At least one of the following" or similar expressions refers to any combination of these items, including any combination of single items or plural items. For example, "at least one of a, b, or c", or "at least one of a, b, and c" can all mean: a, b, c, ab (i.e., a and b), ac, bc, or abc, where a, b, c can be single or multiple, respectively.

[0016] It should be understood that in the various embodiments of the present application, the size of the serial numbers of the above-mentioned processes does not mean the order of execution, some or all of the steps can be executed in parallel or sequentially, and the execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the present application.

[0017] The terms used in the embodiments of the present application are only for the purpose of describing specific embodiments, and are not intended to limit the present application. The singular forms "a", "said" and "the" used in the embodiments of the present application and the appended claims are also intended to include plural forms, unless the context clearly indicates other meanings.

[0018] The weight of the relevant components mentioned in the embodiment description of the present application can not only refer to the specific content of each component, but also represent the proportional relationship between the weights of the components. Therefore, as long as the content of the relevant components is proportionally enlarged or reduced according to the embodiment description of the present application, it is within the scope disclosed in the embodiment description of the present application. Specifically, the mass described in the embodiment description of the present application can be a mass unit known in the chemical industry such as μg, mg, g, kg, etc.

[0019] The terms "first" and "second" are used only for descriptive purposes to distinguish objects such as substances from each other, and should not be understood as indicating or implying relative importance or implicitly indicating the number of indicated technical features. For example, without departing from the scope of the embodiments of the present application, the first XX may also be referred to as the second XX, and similarly, the second XX may also be referred to as the first XX. Thus, features defined as "first" and "second" may explicitly or implicitly include one or more of the features.

[0020] The term "pH" is an abbreviation of "Pondus Hydrogenii", which means acidity or alkalinity, and describes the degree of acidity or alkalinity of an aqueous solution.

[0021] The term "Calvin cycle" is also known as the photosynthetic carbon cycle (carbon reaction). It is a metabolic process that allows animal matter to regenerate after entering and leaving the cycle in the form of molecules. Carbon enters the Calvin cycle in the form of carbon dioxide and leaves the Calvin cycle in the form of sugar. The entire cycle uses adenosine triphosphate (ATP) as an energy source and consumes reduced form nicotinamide adenine dinucleotide phosphate (NADPH) in a reduced energy level, which can increase high-energy electrons to produce sugar.

[0022] The term "SCCM" is an abbreviation for "Standard cubic centimeter per minute", which is a unit of volume flow rate, representing standard milliliters per minute.

[0023] The term "U" is the unit of enzyme activity, the rate at which a unit amount of enzyme produces a unit of substrate per unit time.

[0024] In the current method of synthesizing glucose by hydrolyzing starch, a two-step method is mainly used to hydrolyze starch to produce glucose, including two steps of high-temperature liquefaction and high-temperature saccharification of starch. The key enzymes used in these two steps are mainly α-amylase and saccharifying enzyme. In the first step of high-temperature liquefaction, the starch is first heated at high temperature for gelatinization, and then a heat-resistant α-amylase is added to quickly liquefy into dextrin and small molecular oligosaccharides under high temperature conditions of pH 6.0-6.5 and 95°C-105°C; in the second step of high-temperature saccharification, the hydrolyzed gelatinized liquid is cooled to about 60°C-65°C, and after adjusting the pH to 4.0-4.5, a saccharifying enzyme derived from Aspergillus niger is added thereto, and then dextrin and small molecular oligosaccharides are further hydrolyzed into glucose. It can be seen that starch requires high-temperature cooking and continuous heating during gelatinization, liquefaction and hydrolysis saccharification, which consumes a lot of energy, increases equipment investment, and greatly increases production costs. At the same time, the optimal pH for saccharifying enzymes and α-amylases is also different, and the pH needs to be adjusted repeatedly, which not only wastes reagents but also pollutes the environment.

[0025] In the method of biotransformation to synthesize glucose, it mainly relies on plants, algae and certain microorganisms to carry out photosynthesis, using light energy to convert water and carbon dioxide into glucose. The process of photosynthesis is divided into light reaction and dark reaction, and the overall reaction formula is: 6CO2+6H2O→C6H 12 O6+6O2. Specifically, in the light reaction, the photosynthetic systems 1 and 2 of the plant absorb photons of 700nm and 680nm, and transfer the electrons obtained by photolysis of water molecules to nicotinamide adenine dinucleotide phosphate (NADP + ), generating NADPH. At the same time, the hydrogen ions produced by water photolysis move outward from the thylakoid through the protein complex of the thylakoid membrane, releasing potential energy, which is used to synthesize adenosine triphosphate (ATP) for use in the dark reaction. In the dark reaction, the dark reaction includes three stages: carboxylation, reduction, and regeneration of ribulose diphosphate. Most plants integrate the absorbed CO2 into the five-carbon sugar molecule ribulose-1,5-bisphosphate (RuBP) through ribulose-1,5-bisphosphate carboxylase / oxygenase (RuBisCO) to form an unstable six-carbon compound, which is then decomposed into two three-carbon compounds 3-phosphoglycerate (3-PGA). These compounds are converted into NADPH and H generated by the light reaction. +Reduction, consumption of ATP, and generation of glyceraldehyde 3-phosphate (G3P). G3P is converted into ketose dihydroxyacetone phosphate (DHAP) by triose phosphate isomerase, and G3P and DHAP are condensed by fructose 1,6-bisphosphate aldolase (FBA) to form fructose-1,6-bisphosphate (FBP). It is then hydrolyzed by fructose bisphosphatase (FBPase) to form fructose-6-phosphate (F6P), which is then isomerized to glucose-6-phosphate (G6P) by phosphoglucose isomerase (PGI), and further hydrolyzed by glucose-6-phosphatase (G6Pase) to form glucose. One carbon atom is used to synthesize glucose, and the remaining five carbon atoms are regenerated into RuBP, and the cycle continues. This process is also called the Calvin cycle. One molecule of glucose can be generated every six Calvin cycles.

[0026] In actual sugar production engineering, the main method of biological conversion is to extract glucose after biological fermentation. During the fermentation process, microorganisms convert carbon sources into glucose through metabolic pathways. After the fermentation is completed, glucose is extracted and purified by physical and biochemical methods such as filtration, centrifugation, and chromatography. The process of photosynthesis is essentially a continuous process of a series of energy conversion and chemical reactions, including the absorption of solar energy, the subsequent conversion of light energy to electrical energy, and then the conversion of electrical energy into chemical energy, and finally fixed as stable chemical energy. However, this process is not completely efficient. Only a part of the solar spectrum can be absorbed, and most of the light energy will be lost in the process due to factors such as light reflection and transmission, photoelectric conversion efficiency, and thermodynamic limitations. Theoretically, the maximum photosynthesis conversion efficiency of C4 plants is only 6%, and that of C3 plants is even lower, only 4.6%. In addition, environmental factors such as weather changes and soil conditions will also affect photosynthesis, making the actual photosynthesis conversion efficiency usually only about 1%. Furthermore, due to the need to cultivate organisms, the production cycle is long.

[0027] The embodiment of the present application provides a method for synthesizing glucose by photoenzyme cascade catalysis, comprising the following steps: subjecting an oxidized coenzyme to a photocatalytic reaction to obtain a reduced coenzyme. Using a carbon dioxide source and a pentose compound as raw materials, a first series of enzyme catalytic reactions are performed, and during the first series of enzyme catalytic reactions, the reduced coenzyme is used as a substrate to obtain glyceraldehyde 3-phosphate and ketose dihydroxyacetone phosphate. Using glyceraldehyde 3-phosphate and ketose dihydroxyacetone phosphate as raw materials, a second series of enzyme catalytic reactions are performed to obtain glucose.

[0028] Enzymes are proteins or RNAs produced by living cells that are highly specific and catalytically efficient for their substrates. Enzymes have a unique three-dimensional structure, and their active sites have a specific shape to bind to the corresponding substrates. The shape, charge distribution, and hydrophobic / hydrophilic properties of the active sites complement those of the substrates, allowing for precise fit.

[0029] In the embodiment of the present application, the oxidized coenzyme is converted into the reduced coenzyme by photocatalysis, and the carbon dioxide provided by the carbon dioxide source is fixed to the pentose compound under the action of enzyme catalysis, and is converted into glyceraldehyde 3-phosphate and ketose dihydroxyacetone phosphate through a series of enzyme catalytic reactions, and the reduced coenzyme obtained by photocatalysis is used as a substrate in the enzyme catalytic reaction. Glucose is then converted by condensing, hydrolyzing, isomerizing and other steps of glyceraldehyde 3-phosphate and ketose dihydroxyacetone phosphate under the action of enzyme catalysis.

[0030] The method for synthesizing glucose by photoenzyme cascade catalysis provided in the present application utilizes photocatalytic technology to generate reduced coenzymes, and the generated reduced coenzymes are combined with a variety of other enzymes to carry out a series of enzyme-catalyzed reactions, thereby realizing the photoenzyme coupling catalysis synthesis of glucose. Since each enzyme is selective for the substrates and products of its reaction, it ensures that a series of enzyme-catalyzed reaction processes are highly regulated, thereby realizing the efficient and directional selective conversion of carbon dioxide to glucose. The present application combines the two green process technologies of photocatalysis and enzyme catalysis. The reaction rates of photocatalysis and enzyme catalysis are both at the millisecond level, and artificial synthesis does not require the cultivation of organisms, nor is it affected by weather, soil, etc., which greatly shortens the production cycle and opens up a new path for the green synthesis technology of glucose.

[0031] In some embodiments, the oxidized coenzyme is subjected to a photocatalytic reaction in the presence of a photocatalyst and an electronic intermediate.

[0032] Photocatalysts are a class of chemical substances that can promote chemical reactions under light conditions. They can accelerate chemical reactions without participating in the reactions themselves. Photocatalysts can catalyze when excited by photons. Photocatalysts usually have a special electronic band structure that can absorb light energy and generate electron-hole pairs. These electrons and holes participate in redox reactions, thereby driving the chemical reactions.

[0033] Electron intermediates refer to temporary compounds or molecular forms generated during a series of chemical reactions, also known as electron mediators. When these electron intermediates obtain electrons and hydrogen ions (protons, H + ) becomes more stable and can more easily transfer these electrons to another molecule, that is, it has a stronger reducing ability.

[0034] Photocatalysts are excited to generate electrons under the action of photocatalysis for use by electron intermediates. The electron intermediates that obtain electrons combine with hydrogen ions ionized by water, thus having strong reducing ability, and transfer electrons and hydrogen ions to oxidized coenzymes to form reduced coenzymes. The reduced coenzymes obtained can provide reducing ability in subsequent enzyme-catalyzed reactions. The electron intermediates and enzymes in the reaction have structural specificity and can provide specific sites, greatly improving product selectivity.

[0035] In some embodiments, the electronic intermediate comprises [Cp*Rh(bpy)(H2O)] 2+ 、Cp*Rh(phen)Cl、Ru(bpy)3 2+ , methyl viologen.

[0036] All of the above substances can store and transfer electrons by changing the oxidation state of the central metal or the charge state of the molecule itself. In redox reactions, they can be reduced or oxidized, thereby transferring electrons between reactants and products. Therefore, they can all act as electron intermediates in photocatalytic reactions and play a role in electron transfer.

[0037] In this embodiment, the electronic intermediate is [Cp*Rh(bpy)(H2O)] 2+ [Cp*Rh(bpy)(H2O)] 2+ It has higher reaction efficiency and selectivity in photocatalytic reaction, thus shortening the whole glucose production cycle and improving the yield.

[0038] [Cp*Rh(bpy)(H2O)] 2+ The specific reaction process is: [Cp*Rh(bpy)(H2O)] 2+ It gains two electrons and loses a water molecule to convert into Cp*Rh(bpy), and combines with the hydrogen ion (H + ), generating rhodium hydride [Cp*Rh(bpy)H] + . [Cp*Rh(bpy)H] + It has strong reducing ability and directly transfers electrons and hydrogen ions to coenzymes.

[0039] In some embodiments, the oxidized coenzyme includes NAD + and NADP + At least one of .

[0040] NAD + Nicotinamide adenine dinucleotide is an oxidized form of coenzyme I. + It is reduced to form NADH. NADH is the reduced form of nicotinamide adenine dinucleotide, also known as reduced coenzyme I.

[0041] NADP + Nicotinamide adenine dinucleotide phosphate is titanium oxide, also known as oxidized coenzyme II. In the photocatalytic reaction, NADP + It is reduced to form NADPH. NADPH is the reduced form of nicotinamide adenine dinucleotide phosphate, also known as reduced coenzyme II.

[0042] In this embodiment, the oxidized coenzyme is NAD + .NAD + Compared with NADP + It has the advantage of low price and can reduce production costs.

[0043] It should be added that when the oxidized coenzyme includes NAD + When the wavelength is less than 400 nanometers (such as ultraviolet light), it may damage NADH.

[0044] In some embodiments, the photocatalyst comprises a semiconductor photocatalyst.

[0045] Semiconductor photocatalysts use the electronic properties of semiconductor materials to accelerate chemical reactions. By absorbing light, semiconductor photocatalysts can excite electrons to jump from the valence band to the conduction band, forming electron-hole pairs. These electrons and holes can participate in redox reactions, thereby reducing the activation energy of the reaction and accelerating the reaction rate.

[0046] In some embodiments, the semiconductor photocatalyst includes at least one of a carbon nitride catalyst, a cadmium sulfide catalyst, a molybdenum disulfide catalyst, an iron trioxide catalyst, a metal organic framework catalyst, and a covalent organic framework catalyst. The semiconductor photocatalyst can be appropriately selected according to the wavelength range of the light. For example, the excitation wavelength of the carbon nitride catalyst is generally 390 to 640 nanometers, and visible light can be selected for irradiation; the excitation wavelength of the cadmium sulfide catalyst is between 450 and 620 nanometers, and visible light can be selected for irradiation.

[0047] Therefore, in the embodiment of the present application, the wavelength range of the illumination is ≥400 nanometers, and the spectral range of the light energy is wider than that of chlorophyll.

[0048] In some embodiments, the carbon nitride catalyst in the semiconductor photocatalyst is graphite carbon nitride. Graphite carbon nitride (g-C3N4) has excellent thermal stability and chemical stability. In addition, the band gap of g-C3N4 is suitable for visible light driving. It is worth mentioning that g-C3N4 has a rich aromatic hydrocarbon structure, which enables it to effectively react with [Cp*Rh(bpy)(H2O)] 2+ and NAD + Electronic transmission.

[0049] In some embodiments, the carbon nitride catalyst in the semiconductor photocatalyst is prepared by calcining a carbon nitrogen compound as a precursor.

[0050] The carbon-nitrogen compound includes at least one of melamine, dicyandiamide and urea. Melamine, dicyandiamide and urea can all be used to obtain g-C3N4 by calcination.

[0051] In some embodiments, the calcination temperature is 450°C to 600°C.

[0052] The calcination temperature may be 450°C, 475°C, 500°C, 525°C, 550°C, 575°C, 600°C, etc., or any value between the adjacent values ​​above.

[0053] In some embodiments, the calcination time is 2 to 4 hours.

[0054] The calcination time can be 2 hours, 2.5 hours, 3 hours, 3.5 hours, 4 hours, etc., or any value between the adjacent values ​​above.

[0055] In some embodiments, the heating rate of calcination is 2° C. / min to 8° C. / min.

[0056] The heating rate of calcination can be 2°C / min, 3°C / min, 4°C / min, 5°C / min, 6°C / min, 7°C / min, 8°C / min, etc., or any value between the above adjacent values.

[0057] In the calcination reaction, the factors of temperature, time and heating rate will affect the structure and reducing ability of the photocatalyst product. Experiments have found that the above temperature range, time and heating rate can control the obtained photocatalyst to have suitable physical and chemical properties and photocatalytic activity.

[0058] In some embodiments, the pH of the photocatalytic reaction system is 6.0 to 9.0. In the photocatalytic reaction, different pH conditions may affect the activity of the photocatalyst, the electron transfer ability of the electron intermediate, etc. By controlling the pH value of the photocatalytic reaction system between 6.0 and 9.0, the generation effect of the reduced coenzyme can be ensured.

[0059] The pH of the photocatalytic reaction system can be 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, etc., or any value between the adjacent values ​​above.

[0060] In some embodiments, in the first series of enzyme-catalyzed reactions, the reduced coenzyme is oxidized to an oxidized coenzyme.

[0061] In the photocatalytic reaction, the oxidized coenzyme serves as the reaction substrate and the reduced coenzyme serves as the reaction product; while in the first series of enzyme-catalyzed reactions, the reduced coenzyme serves as the reaction substrate and the oxidized coenzyme serves as the reaction product, thereby achieving cyclic regeneration between the oxidized coenzyme and the reduced coenzyme.

[0062] Combination Figure 1As shown, the method for synthesizing glucose by photoenzyme cascade catalysis provided in the embodiment of the present application includes a light reaction and a dark reaction: the light reaction uses photocatalytic technology to generate reduced coenzymes, and the dark reaction uses a series of enzyme catalytic reactions to generate glucose by combining the reduced coenzymes generated by light with other enzymes, thereby realizing the photoenzyme coupling catalytic synthesis of glucose. Among them, the light reaction can photocatalyze the oxidized coenzyme to generate reduced coenzymes under the conditions of a photocatalyst and an electron intermediate, and the dark reaction uses a carbon dioxide source and a pentose compound as raw materials, and obtains a glucose reaction after a first series of enzyme catalytic reactions and a second series of enzyme catalytic reactions. In some embodiments, the first series of enzyme catalytic reactions includes the following steps: a carbon dioxide source reacts with a pentose compound (C5) under the action of a first enzyme to form 3-phosphoglyceric acid (3-PGA). 3-phosphoglyceric acid forms phosphoglyceric acid (1,3-BPGA) under the action of a second enzyme and nucleoside triphosphates. Phosphoglyceric acid forms glyceraldehyde 3-phosphate (G3P) under the action of reduced coenzymes and a third enzyme. Part of the glyceraldehyde 3-phosphate forms ketose dihydroxyacetone phosphate (DHAP) under the action of a fourth enzyme.

[0063] Combination Figure 1 As shown, in some embodiments, the first enzyme includes ribulose-1,5-bisphosphate carboxylase / oxygenase (RuBisCO). The second enzyme includes phosphoglycerate kinase (PGK). The third enzyme includes glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The fourth enzyme includes triosephosphate isomerase (TPI). Nucleoside triphosphate includes adenosine triphosphate (ATP).

[0064] In some embodiments, the carbon dioxide source includes at least one of carbon dioxide, potassium bicarbonate, and sodium bicarbonate.

[0065] In some embodiments, the pentose compound comprises ribulose-1,5-bisphosphate (RuBP).

[0066] RuBisCO enzyme catalyzes the carboxylation reaction of CO2 and RuBP, adding CO2 to the second carbon atom of RuBP to generate an unstable 6-carbon sugar acid intermediate, which rapidly splits into two 3-phosphoglycerate (3-PGA) molecules.

[0067] PGK catalyzes the conversion of 3-PGA to 1,3-BPGA, and in the process ATP is consumed and converted into ADP.

[0068] GAPDH catalyzes the reduction of 1,3-BPGA by the reduced coenzyme NADH to generate G3P, and NADH is oxidized to NAD +, the reaction is a redox reaction, in which NADH acts as a reducing agent, transferring electrons to 1,3-BPGA, reducing it to G3P. GAPDH acts as a catalyst in the reaction, using the reducing power of NADH to drive the reaction.

[0069] G3P is converted into DHAP under the catalysis of TPI. This reaction is an isomerization reaction, which transfers the 3-carbon hydroxyl group (-OH) of G3P from C3 to C2 to generate DHAP, providing preparation for the subsequent synthesis of hexose compounds.

[0070] Combination Figure 1 As shown, in some embodiments, the second series of enzyme-catalyzed reactions includes the following steps: glyceraldehyde 3-phosphate and ketose dihydroxyacetone phosphate form fructose-1,6-bisphosphate (FBP) under the action of the fifth enzyme. Fructose-1,6-bisphosphate forms fructose-6-phosphate (F6P) under the action of the sixth enzyme. Fructose-6-phosphate forms glucose-6-phosphate (G6P) under the action of the seventh enzyme. Glucose-6-phosphate forms glucose under the action of the eighth enzyme.

[0071] Combination Figure 1 As shown, in some embodiments, the fifth enzyme comprises fructose 1,6-bisphosphate aldolase (FBA).

[0072] The sixth enzyme includes fructose bisphosphatase (FBPase). The seventh enzyme includes phosphoglucose isomerase (PGI).

[0073] The eighth enzyme includes glucose-6-phosphatase (G6Pase).

[0074] In the second series of enzyme-catalyzed reactions, it can be reversed through the structure of glucose. We know that in the process of glycolysis, G6P is the starting material of glycolysis, and is generated by phosphorylation reaction of glucose and phosphate under the catalysis of G6Pase. Therefore, it can be reversed to use G6P to convert into glucose. G6P can be obtained by the catalytic reaction of F6P through PGI. F6P can be obtained by the hydrolysis reaction of FBP through FBPase. FBP is composed of two trisaccharide units, namely G3P and DHAP, and G3P and DHAP are synthesized into FBP through FBA catalysis.

[0075] The following describes the invention in conjunction with specific embodiments.

[0076] Example 1

[0077] A method for synthesizing glucose by photoenzyme cascade catalysis is prepared by the following steps:

[0078] S1. Preparation of photocatalyst: Using melamine as a precursor, 5 g of melamine was placed in a quartz boat, which was then placed in a muffle furnace. In an air atmosphere, the melamine was calcined at 500° C. for 2 hours at a heating rate of 5° C. / min to obtain g-C3N4 nanosheets.

[0079] S2, photocatalytic regeneration of NADH: In a quartz reactor, 3 mg / mL of the photocatalyst (g-C3N4 nanosheets) prepared in step S1, 0.25 mM of the electronic intermediate ([Cp*Rh(bpy)(H2O)] 2+ ), 1mM oxidized coenzyme (NAD + ), 15 w / v% triethanolamine and 0.1 M phosphate buffer were added to the reaction system, and NADH was obtained by irradiation reaction using a xenon lamp with a 400 nm bandpass filter. The pH of the reaction system was 8.0. Before the reaction, argon gas was introduced at a flow rate of 30 SCCM.

[0080] S3, photoenzyme cascade synthesis of glucose: 0.5mM pentose compound (RuBP), 1mM nucleoside triphosphate (ATP), and NADH obtained in step S2 were added to 5mM magnesium chloride (MgCl2), 66mM potassium bicarbonate (KHCO3), and 5mM dithiothreitol solution. Then, 0.0125U ribulose-1,5-bisphosphate carboxylase / oxygenase, 10U phosphoglycerate kinase, 5U glyceraldehyde-3-phosphate dehydrogenase, 5U triosephosphate isomerase, 10U fructose-bisphosphate aldolase, 10U fructose-1,6-phosphatase, and 10U phosphoglucose isomerase were further added. Finally, carbon dioxide gas was introduced into the solution to react and obtain glucose.

[0081] Detection and analysis

[0082] The product in step S2 was detected by UV-visible spectrophotometer. The absorption peak of the product NADH was 340 nm, and the wavelength was 340 nm and the molar absorption coefficient was 6220 M. -1 cm -1 Calculate the concentration of generated NADH. Figure 2 shown. Figure 2 The UV-visible absorption spectrum of the solution before the photocatalytic reaction and the UV-visible absorption spectrum of the solution after 40 minutes of photocatalytic reaction are shown in Figure 2. It can be seen that at 340nm, the concentration after 40 minutes of reaction is higher than that before the reaction. It is calculated that the concentration of NADH can reach 0.78mmol / L and the conversion rate is 78%. By calculating the concentration of NADH, the yield of G3P can also be measured to be 1.6mmol L -1 h -1 .

[0083] According to the above data combined with the entire reaction route, in the embodiment of the present application, each Calvin cycle fixes a carbon dioxide molecule, and finally generates a G3P through a series of enzyme-catalyzed reactions. Every two Calvin cycles can synthesize a molecule of glucose, that is, two G3Ps synthesize one glucose. The efficiency of synthesizing glucose by artificial photosynthesis (photocatalysis + enzyme catalysis) can be as high as 15%, while the efficiency of synthesizing glucose by natural photosynthesis is 1%. In comparison, the method for synthesizing glucose by photoenzyme cascade catalysis provided in the embodiment of the present application has a significant improvement in synthesis efficiency.

[0084] The above description is only a preferred embodiment of the present application and is not intended to limit the present application. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present application should be included in the protection scope of the present application.

Claims

1. A method for synthesizing glucose by photoenzyme cascade catalysis, characterized in that: The following steps are involved: The oxidized coenzyme is subjected to a photocatalytic reaction to obtain a reduced coenzyme; A first series of enzyme catalytic reactions are carried out using a carbon dioxide source and a pentose compound as raw materials, and the reduced coenzyme is used as a substrate during the first series of enzyme catalytic reactions to obtain glyceraldehyde 3-phosphate and ketose dihydroxyacetone phosphate; The glyceraldehyde 3-phosphate and the ketose dihydroxyacetone phosphate are used as raw materials to carry out a second series of enzyme catalytic reactions to obtain glucose.

2. The method for synthesizing glucose by photoenzyme cascade catalysis according to claim 1, characterized in that: The oxidized coenzyme undergoes a photocatalytic reaction under the conditions of a photocatalyst and an electron intermediate.

3. The method for synthesizing glucose by photoenzyme cascade catalysis according to claim 2, characterized in that: The electronic intermediates include [Cp*Rh(bpy)(H2O)] 2+ 、Cp*Rh(phen)Cl、Ru(bpy)3 2+ , at least one of methyl violet chloride; And / or, the photocatalyst comprises a semiconductor photocatalyst.

4. The method for synthesizing glucose by photoenzyme cascade catalysis according to claim 3, characterized in that: The semiconductor photocatalyst includes at least one of a carbon nitride catalyst, a cadmium sulfide catalyst, a molybdenum disulfide catalyst, a ferric oxide catalyst, a metal organic framework catalyst, and a covalent organic framework catalyst.

5. The method for synthesizing glucose by photoenzyme cascade catalysis according to claim 4, characterized in that: The carbon nitride catalyst in the semiconductor photocatalyst is graphite phase carbon nitride; And / or, the carbon nitride catalyst in the semiconductor photocatalyst is prepared by calcining a carbon nitrogen compound as a precursor.

6. The method for synthesizing glucose by photoenzyme cascade catalysis according to any one of claims 1 to 5, characterized in that: In the first series of enzyme-catalyzed reactions, the reduced coenzyme is oxidized to the oxidized coenzyme.

7. The method for synthesizing glucose by photoenzyme cascade catalysis according to any one of claims 1 to 5, characterized in that: The first series of enzyme catalyzed reactions comprises the following steps: The carbon dioxide source reacts with the pentose compound under the action of the first enzyme to form 3-phosphoglyceric acid; The 3-phosphoglycerate forms phosphoglycerate under the action of the second enzyme and nucleoside triphosphate; The phosphoglycerate forms the glyceraldehyde 3-phosphate under the action of the reduced coenzyme and the third enzyme; Part of the glyceraldehyde 3-phosphate forms the ketose dihydroxyacetone phosphate under the action of the fourth enzyme.

8. The method for synthesizing glucose by photoenzyme cascade catalysis according to claim 7, characterized in that: The first enzyme comprises ribulose-1,5-bisphosphate carboxylase / oxygenase; and / or, The second enzyme comprises phosphoglycerate kinase; and / or, The third enzyme comprises glyceraldehyde-3-phosphate dehydrogenase; and / or, The fourth enzyme includes triose phosphate isomerase.

9. The method for synthesizing glucose by photoenzyme cascade catalysis according to any one of claims 1 to 5, characterized in that: The second series of enzyme catalyzed reactions comprises the following steps: The glyceraldehyde 3-phosphate and the ketose dihydroxyacetone phosphate form fructose-1,6-bisphosphate under the action of the fifth enzyme; The fructose-1,6-diphosphate is converted into fructose-6-phosphate under the action of the sixth enzyme; The fructose-6-phosphate is converted into glucose-6-phosphate under the action of the seventh enzyme; The glucose-6-phosphate is converted into the glucose under the action of the eighth enzyme.

10. The method for synthesizing glucose by photoenzyme cascade catalysis according to claim 9, characterized in that: The fifth enzyme comprises fructose 1,6-bisphosphate aldolase; and / or, The sixth enzyme comprises fructose bisphosphatase; and / or, The seventh enzyme includes phosphoglucose isomerase; and / or, The eighth enzyme includes glucose-6-phosphatase.

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