A method for enzymatically catalyzing and utilizing starch or its derivatives

Abstract

The present invention discloses an enzymatic catalytic utilization method of starch or its derivatives, and belongs to the field of enzyme catalysis. The present invention uses starch and its derivatives as substrates, and in a multi-enzyme reaction system, the substrates are efficiently converted into high value-added products, including inositol, tagatose, mannose, psicose, and fructose 1,6-diphosphate, by an in vitro multi-enzyme molecular machine. The present invention realizes the high value-added utilization of starch and its derivatives by adding an enzyme that can utilize the by-product glucose, significantly improves the conversion rate of raw materials, has the advantages of high product yield, cheap raw materials, simple operation, low production cost, etc., and can realize large-scale production of products.

Description

Technical Field

[0001] The present invention relates to a method for enzymatically catalyzing the utilization of starch or its derivatives, belonging to the field of enzyme catalysis. Background Art

[0002] Starch is an inexpensive biological raw material, which can be extracted from starch-containing substances such as corn, sweet potato, wheat, wild acorns, and kudzu root. Besides being used for food, starch is mainly used in industry to prepare dextrin, maltose, glucose, alcohol, etc., and is also used for preparing printing paste, sizing textiles, sizing paper, pressing pharmaceutical tablets, etc. Preparing products with higher added value from starch, such as inositol, rare sugars, fructose 1,6-diphosphate, etc., has more extensive application value.

[0003] Methods for preparing glucose 6-phosphate using starch and its derivatives have been reported. It mainly uses the combination of glycogen phosphorylase (EC 2.4.1.1) and phosphoglucomutase (EC 5.4.2.2). However, due to the reaction equilibrium, the conversion rate is only about 40% (Zhou W, You C, Ma H, Ma Y, Zhang YH. 2016. One-Pot Biosynthesis of High-Concentration alpha-Glucose 1-Phosphate from Starch by Sequential Addition of Three Hyperthermophilic Enzymes. J Agric Food Chem 64(8):1777-83.). Methods for improving the conversion rate include coupling the use of 4-α-glucan transferase (4GT, EC 2.4.1.25) and polyphosphate glucokinase (EC 2.7.1.63) in the later stage of the reaction. However, since 4GT can only be added in the later stage of the reaction, and the activities of the enzymes added at the beginning of the reaction (glycogen phosphorylase and phosphoglucomutase) decrease significantly, resulting in an extended overall reaction time and a low conversion rate. Summary of the Invention

[0004] The present invention provides a method for utilizing starch and / or starch derivatives, and the method comprises the following reaction steps:

[0005] (1) Using β-amylase (EC 3.2.1.2) for catalysis to convert the substrate into maltose, and the substrate is starch and / or starch derivatives;

[0006] (2) Catalyzed by maltose phosphorylase (EC 2.4.1.8), maltose and inorganic phosphate are converted into β-D-glucose 1-phosphate and glucose;

[0007] (3) Catalyzed by β-glucose phosphomutase (EC 5.4.2.6), β-D-glucose 1-phosphate is converted into β-D-glucose 6-phosphate;

[0008] (4) Catalyzed by polyphosphate glucokinase (EC 2.7.1.63), the glucose and polyphosphate in step (2) are converted into α-D-glucose 6-phosphate;

[0009] Steps (1)-(4) are carried out by a "one-pot method" to obtain the product.

[0010] In one embodiment, the method includes establishing a multi-enzyme reaction system with starch and / or starch derivatives as substrates and carrying out an enzymatic hydrolysis reaction. The multi-enzyme reaction system further includes inorganic phosphate, β-amylase, maltose phosphorylase, β-glucose phosphomutase, polyphosphate glucokinase, and polyphosphate.

[0011] In one embodiment, the starch of the present invention is soluble starch, including but not limited to corn starch, wheat starch, tapioca starch, potato starch, or other starch types known in the art; the starch derivatives are selected from any one or more of partially hydrolyzed starch, starch dextrin, maltodextrin, maltopolysaccharide, or other modified starches known in the art.

[0012] In one embodiment, the polyphosphate is sodium polyphosphate, such as one of sodium tripolyphosphate salt and sodium hexametaphosphate salt, or two of them in any ratio, preferably sodium hexametaphosphate.

[0013] In one embodiment, the inorganic phosphate is selected from inorganic phosphates, such as any one of sodium phosphate, sodium dihydrogen phosphate, disodium hydrogen phosphate, potassium phosphate, potassium dihydrogen phosphate, and dipotassium hydrogen phosphate. In another embodiment, the inorganic phosphate is derived from a phosphate buffer solution.

[0014] According to the present invention, in the reaction system of the present invention, the amounts of the substrate or enzyme are not particularly limited, and the reaction of the substrate can be catalyzed to generate the corresponding product to achieve the object of the present invention. In one embodiment, based on the total volume of the reaction system, the concentration of the substrate is 1-800 g / L, such as 20-200 g / L; the concentration of polyphosphate is 1-500 mM, such as 20-200 mM; β-amylase is 0.1-10000 U / mL, such as 1-1000 U / mL; maltose phosphorylase is 0.1-10000 U / mL, such as 1-1000 U / mL; the amount of β-glucose phosphomutase is 0.1-10000 U / mL, such as 1-1000 U / mL; the amount of polyphosphate glucokinase is 0.1-10000 U / mL, such as 1-1000 U / mL; the concentration of inorganic phosphorus is 5-500 mM; more preferably, the concentration of inorganic phosphorus is 10-50 mM.

[0015] In one embodiment, glucose 6-phosphate, such as α-D-glucose 6-phosphate and / or β-D-glucose 6-phosphate, is obtained by the above method.

[0016] According to the present invention, the method further comprises step (5): α-D-glucose 6-phosphate and / or β-D-glucose 6-phosphate further undergoes an enzymatic reaction to obtain a final product, and the final product is at least one of inositol, tagatose, allulose, mannose or fructose 1,6-diphosphate. In a specific embodiment, step (5) includes any one of the following steps:

[0017] (5a) Adding inositol-1-phophate synthase (EC 5.5.1.4) and inositol monophosphatase (EC 3.1.3.25) to obtain inositol;

[0018] (5b) Adding phosphoglucose isomerase (EC 5.3.1.9), tagatose 6-phosphate 4-epimerase (EC 5.1.3.40) and phosphatase to obtain tagatose;

[0019] (5c) Adding phosphoglucose isomerase (EC 5.3.1.9), D-allulose 6-phosphate 3-epimerase (EC 5.1.3.-) and phosphatase to obtain allulose;

[0020] (5d) Add phosphoglucose isomerase (EC 5.3.1.9), mannose 6-phosphate isomerase (EC 5.3.1.8) and phosphatase to obtain mannose; or

[0021] (5e) Add pyrophosphate, phosphoglucose isomerase (EC 5.3.1.9) and pyrophosphate-dependent phosphofructokinase (EC 2.7.1.90) to obtain fructose 1,6-diphosphate.

[0022] In one embodiment, steps (1)-(5) are carried out by a "one-pot method".

[0023] In one embodiment, in step (5), based on the total volume of the reaction system, the dosages of inositol 1-phosphate synthase, inositol monophosphatase, phosphoglucose isomerase, tagatose 6-phosphate 4-epimerase, psicose 6-phosphate 3-epimerase, mannose 6-phosphate isomerase, pyrophosphate-dependent phosphofructokinase and phosphatase are 0.1-10000 U / mL, such as 1-1000 U / mL, and further such as 2-100 U / mL.

[0024] The phosphatase described in the present invention can be any one of phytase, acid phosphatase, alkaline phosphatase or specific phosphatase; preferably at least one of tagatose 6-phosphate specific phosphatase, psicose 6-phosphate specific phosphatase or mannose 6-phosphate specific phosphatase.

[0025] According to the present invention, in the reaction system of the reaction, it may further include any one or more of a buffer solution and / or a magnesium salt. Those skilled in the art can understand that various buffer solutions can be used in the present invention, such as HEPES buffer solution, Tris-HCl buffer solution, MOPS buffer solution, citrate buffer solution, phosphate buffer solution, etc.; the pH of the buffer solution is 4.0-10.0, and the concentration of the buffer solution is 5-500 mM; more preferably, the concentration of the buffer solution is 10 mM and the pH is 6.5-7.5, such as 7.0. In the reaction system of the present invention, a magnesium salt may also be included, and various magnesium salts can be used in the present invention, such as magnesium chloride, magnesium sulfate, etc.; preferably the magnesium salt is magnesium sulfate, and the concentration is 0.01-500 mM; more preferably, the concentration of magnesium sulfate is 20-25 mM.

[0026] According to the present invention, in the reaction system of the reaction, a debranching enzyme may also be added, or the starch or starch derivative is pretreated with a debranching enzyme before step (1). In one embodiment, the debranching enzyme is any one or both of isoamylase (EC 3.2.1.68) or pullulanase (EC 3.2.1.41). The dosage of the debranching enzyme is based on the total volume of the reaction system, and the concentration of the debranching enzyme is 0.1-10000 U / mL, such as 1-1000 U / mL, and further such as 2-100 U / mL.

[0027] According to the present invention, the reaction temperature of the reaction is carried out at 30-90 °C, such as 50 °C; the reaction time is 1-200 hours, such as 24-72 hours.

[0028] In the present invention, the "one-pot method" means that steps (1)-(4) are carried out simultaneously, or steps (1)-(5) are carried out simultaneously. For example, the substrate starch or starch derivative, β-amylase, maltose phosphorylase, β-phosphoglucomutase, polyphosphate glucokinase and polyphosphate are added to the reaction system for reaction, and further one or more of inositol 1-phosphate synthase, inositol monophosphatase, phosphoglucose isomerase, tagatose 6-phosphate 4-epimerase, psicose 6-phosphate 3-epimerase, mannose 6-phosphate isomerase, pyrophosphate-dependent phosphofructokinase and phosphatase are added to construct a multi-enzyme reaction system for reaction.

[0029] According to the above method, the present invention further provides a method for preparing a series of high-value-added products.

[0030] As a specific embodiment, the present invention provides a method for preparing inositol by multi-enzyme catalysis of starch and / or starch derivative in vitro:

[0031] In a multi-enzyme system, using starch and / or starch derivative and polyphosphate as substrates, β-amylase (β-amylase, EC 3.2.1.2), maltose phosphorylase (maltose phosphorylase, EC 2.4.1.8), β-phosphoglucomutase (β-phosphoglucomutase, EC 5.4.2.6), polyphosphate glucokinase (polyphosphate glucokinase, EC2.7.1.63), inositol 1-phosphate synthase (Inositol-1-phophatesynthase, EC 5.5.1.4) and inositol monophosphatase (inositol monophosphatase, EC 3.1.3.25) are added to establish a multi-enzyme system for enzymatic reaction.

[0032] In one embodiment, the concentration of starch and / or starch derivative is 1 - 800 g / L, the concentration of polyphosphate is 1 - 500 mM, the dosage of β - amylase is 0.1 - 10000 U / mL, the dosage of maltose phosphorylase is 0.1 - 10000 U / mL, the dosage of β - glucosephosphate mutase is 0.1 - 10000 U / mL, the dosage of polyphosphate glucokinase is 0.1 - 10000 U / mL, the dosage of inositol 1 - phosphate synthase is 0.1 - 10000 U / mL, and the dosage of inositol monophosphatase is 0.1 - 10000 U / mL.

[0033] Preferably, the dosage of starch and / or starch derivative is 20 g / L, the dosage of polyphosphate is 20 mM, the dosage of β - amylase is 2 U / mL, the dosage of maltose phosphorylase is 2 U / mL, the dosage of β - glucosephosphate mutase is 2 U / mL, the dosage of polyphosphate glucokinase is 2 U / mL, the dosage of inositol 1 - phosphate synthase is 2 U / mL, and the dosage of inositol monophosphatase is 2 U / mL.

[0034] The enzyme - catalyzed reaction system is carried out at 30 - 90 °C for 1 - 200 hours. Preferably, the reaction temperature is 50 °C and the reaction time is 72 hours.

[0035] In the above - mentioned multi - enzyme reaction system, there are also buffer solution, inorganic phosphorus, and magnesium salt. The concentration of inorganic phosphorus is 5 - 500 mM; more preferably, the concentration of inorganic phosphorus is 10 mM. The buffer solution is preferably phosphate buffer solution, the pH of the buffer solution is 4.0 - 10.0, and the concentration of the buffer solution is 5 - 500 mM; more preferably, the concentration of the buffer solution is 10 mM and the pH is 6.5 - 7.0. The magnesium salt is magnesium sulfate, and the concentration is 0.01 - 500 mM; more preferably, the concentration of magnesium sulfate is 20 mM.

[0036] As a specific embodiment, the present invention provides a method for preparing tagatose by multi - enzyme catalysis of starch and / or starch derivative in vitro:

[0037] In a multi-enzyme system, using starch and / or starch derivatives, and polyphosphate as substrates, add β-amylase (β-amylase, EC 3.2.1.2), maltose phosphorylase (maltose phosphorylase, EC 2.4.1.8), β-phosphoglucomutase (β-phosphoglucomutase, EC 5.4.2.6), polyphosphate glucokinase (polyphosphate glucokinase, EC2.7.1.63), phosphoglucose isomerase (Phosphoglucose isomerase, EC 5.3.1.9), tagatose 6-phosphate 4-epimerase (tagatose 6-phosphate 4-epimerase, EC 5.1.3.40), and phosphatase to establish a multi-enzyme reaction system and carry out an enzyme-catalyzed reaction.

[0038] The phosphatase is tagatose 6-phosphate specific phosphatase.

[0039] In a reaction system, the concentration of starch and / or starch derivatives is 1 - 800 g / L, the concentration of polyphosphate is 1 - 500 mM, the dosage of β-amylase is 0.1 - 10000 U / mL, the dosage of maltose phosphorylase is 0.1 - 10000 U / mL, the dosage of β-phosphoglucomutase is 0.1 - 10000 U / mL, the dosage of polyphosphate glucokinase is 0.1 - 10000 U / mL, the dosage of phosphoglucose isomerase is 0.1 - 10000 U / mL, the dosage of tagatose 6-phosphate 4-epimerase is 0.1 - 10000 U / mL, and the dosage of tagatose 6-phosphate phosphatase is 0.1 - 10000 U / mL.

[0040] Preferably, the dosage of starch and / or starch derivatives is 20 g / L, the dosage of polyphosphate is 20 mM, the dosage of β-amylase is 2 U / mL, the dosage of maltose phosphorylase is 2 U / mL, the dosage of β-phosphoglucomutase is 2 U / mL, the dosage of polyphosphate glucokinase is 2 U / mL, the dosage of phosphoglucose isomerase is 2 U / mL, the dosage of tagatose 6-phosphate 4-epimerase is 2 U / mL, and the dosage of tagatose 6-phosphate phosphatase is 2 U / mL.

[0041] The enzyme-catalyzed reaction system is carried out at 30 - 90 °C, and the reaction time is 1 - 200 hours. Preferably, the reaction temperature is 50 °C and the reaction time is 72 hours.

[0042] In the above-mentioned multi-enzyme reaction system, a buffer solution, inorganic phosphate, and magnesium salt are also included. The concentration of inorganic phosphate is 5 - 500 mM; more preferably, the concentration of inorganic phosphate is 10 mM. The buffer solution is preferably a phosphate buffer solution, the pH of the buffer solution is 4.0 - 10.0, and the concentration of the buffer solution is 5 - 500 mM; more preferably, the concentration of the buffer solution is 10 mM and the pH is 6.5 - 7.5. The magnesium salt is magnesium sulfate, and the concentration is 0.01 - 500 mM; more preferably, the concentration of magnesium sulfate is 20 mM.

[0043] As a specific embodiment, the present invention provides a method for preparing allulose from starch and / or starch derivatives by multi-enzyme catalysis in vitro:

[0044] In a multi-enzyme system, using starch and / or starch derivatives, and polyphosphate as substrates, β-amylase (β-amylase, EC 3.2.1.2), maltose phosphorylase (maltose phosphorylase, EC 2.4.1.8), β-phosphoglucomutase (β-phosphoglucomutase, EC 5.4.2.6), polyphosphate glucokinase (polyphosphate glucokinase, EC2.7.1.63), phosphoglucose isomerase (Phosphoglucose isomerase, EC 5.3.1.9), D-allulose 6-phosphate 3-epimerase (D-allulose 6-phosphate 3-epimerase, EC 5.1.3.-), and phosphatase are added to establish a multi-enzyme reaction system for enzymatic catalysis reaction.

[0045] The phosphatase is preferably allulose 6-phosphate specific phosphatase.

[0046] In a reaction system, the concentration of starch and / or starch derivatives is 1 - 800 g / L, the concentration of polyphosphate is 1 - 500 mM, the dosage of β-amylase is 0.1 - 10000 U / mL, the dosage of maltose phosphorylase is 0.1 - 10000 U / mL, the dosage of β-phosphoglucomutase is 0.1 - 10000 U / mL, the dosage of polyphosphate glucokinase is 0.1 - 10000 U / mL, the dosage of phosphoglucose isomerase is 0.1 - 10000 U / mL, the dosage of D-allulose 6-phosphate 3-epimerase is 0.1 - 10000 U / mL, and the dosage of allulose 6-phosphate phosphatase is 0.1 - 10000 U / mL.

[0047] Preferably, the dosage of starch and / or starch derivative is 20 g / L, the dosage of polyphosphate is 20 mM, the dosage of β-amylase is 2 U / mL, the dosage of maltose phosphorylase is 2 U / mL, the dosage of β-phosphoglucomutase is 2 U / mL, the dosage of polyphosphate glucokinase is 2 U / mL, the dosage of phosphoglucose isomerase is 2 U / mL, the dosage of psicose 6-phosphate 3-epimerase is 2 U / mL, and the dosage of psicose 6-phosphate phosphatase is 2 U / mL.

[0048] The enzyme-catalyzed reaction system is carried out at 30-90 °C, and the reaction time is 1-200 hours. Preferably, the reaction temperature is 50 °C and the reaction time is 72 hours.

[0049] In the above multi-enzyme reaction system, a buffer solution, inorganic phosphorus, and magnesium salt are also included. The concentration of inorganic phosphorus is 5-500 mM; more preferably, the concentration of inorganic phosphorus is 10 mM. The buffer solution is preferably a phosphate buffer solution, the pH of the buffer solution is 4.0-10.0, and the concentration of the buffer solution is 5-500 mM; more preferably, the concentration of the buffer solution is 10 mM and the pH is 6.5-7.5. The magnesium salt is magnesium sulfate, and the concentration is 0.01-500 mM; more preferably, the concentration of magnesium sulfate is 20 mM.

[0050] As a specific embodiment, the present invention provides a method for preparing mannose by multi-enzyme catalysis of starch and / or starch derivative in vitro:

[0051] In a multi-enzyme system, using starch and / or starch derivative and polyphosphate as substrates, β-amylase (β-amylase, EC 3.2.1.2), maltose phosphorylase (maltose phosphorylase, EC 2.4.1.8), β-phosphoglucomutase (β-phosphoglucomutase, EC 5.4.2.6), polyphosphate glucokinase (polyphosphate glucokinase, EC2.7.1.63), phosphoglucose isomerase (Phosphoglucoseisomerase, EC 5.3.1.9), mannose 6-phosphate isomerase (mannose 6-phosphate isomerase, EC5.3.1.8), and phosphatase are added to establish a multi-enzyme reaction system for enzyme-catalyzed reaction.

[0052] The phosphatase is preferably mannose 6-phosphate specific phosphatase.

[0053] The phosphoglucose isomerase and mannose 6-phosphate isomerase of the present invention can be replaced by a bifunctional enzyme (phosphoglucose / phosphomannose isomerases, EC 5.3.1.9, EC 5.3.1.8) that simultaneously has phosphoglucose isomerase and mannose 6-phosphate isomerase activities.

[0054] In a reaction system, the concentration of starch and / or starch derivative is 1 - 800 g / L, the concentration of polyphosphate is 1 - 500 mM, the dosage of β-amylase is 0.1 - 10000 U / mL, the dosage of maltose phosphorylase is 0.1 - 10000 U / mL, the dosage of β-glucose phosphomutase is 0.1 - 10000 U / mL, the dosage of polyphosphoglucokinase is 0.1 - 10000 U / mL, the dosage of phosphoglucose isomerase is 0.1 - 10000 U / mL, the dosage of mannose 6-phosphate isomerase is 0.1 - 10000 U / mL, and the dosage of mannose 6-phosphate phosphatase is 0.1 - 10000 U / mL.

[0055] Preferably, the dosage of starch and / or starch derivative is 20 g / L, the dosage of polyphosphate is 20 mM, the dosage of β-amylase is 2 U / mL, the dosage of maltose phosphorylase is 2 U / mL, the dosage of β-glucose phosphomutase is 2 U / mL, the dosage of polyphosphoglucokinase is 2 U / mL, the dosage of phosphoglucose isomerase is 2 U / mL, the dosage of mannose 6-phosphate isomerase is 2 U / mL, and the dosage of mannose 6-phosphate phosphatase is 2 U / mL.

[0056] The enzyme-catalyzed reaction system is carried out at 30 - 90 °C, and the reaction time is 1 - 200 hours. Preferably, the reaction temperature is 50 °C and the reaction time is 72 hours.

[0057] In the above multi-enzyme reaction system, it also contains a buffer solution, inorganic phosphorus, and a magnesium salt. The concentration of inorganic phosphorus is 5 - 500 mM; more preferably, the concentration of inorganic phosphorus is 10 mM. The buffer solution is preferably a phosphate buffer solution, the pH of the buffer solution is 4.0 - 10.0, and the concentration of the buffer solution is 5 - 500 mM; more preferably, the concentration of the buffer solution is 10 mM and the pH is 6.5 - 7.5. The magnesium salt is magnesium sulfate, and the concentration is 0.01 - 500 mM; more preferably, the concentration of magnesium sulfate is 20 mM.

[0058] As a specific embodiment, the present invention provides a method for preparing fructose 1,6-diphosphate from starch and / or starch derivative by multi-enzyme catalysis in vitro:

[0059] In a multi-enzyme system, using starch and / or starch derivatives, as well as polyphosphates and pyrophosphates as substrates, β-amylase (β-amylase, EC 3.2.1.2), maltose phosphorylase (maltose phosphorylase, EC 2.4.1.8), β-phosphoglucomutase (β-phosphoglucomutase, EC 5.4.2.6), polyphosphate glucokinase (polyphosphate glucokinase, EC 2.7.1.63), phosphoglucose isomerase (Phosphoglucose isomerase, EC 5.3.1.9) and pyrophosphate-dependent phosphofructokinase (6-phosphofructokinase, EC 2.7.1.90) are added to establish a multi-enzyme reaction system for enzymatic catalysis reaction.

[0060] In a reaction system, the concentration of the starch and / or starch derivatives is 1 - 800 g / L, the concentration of polyphosphates is 1 - 500 mM, the concentration of pyrophosphates is 1 - 1000 mM, the dosage of β-amylase is 0.1 - 10000 U / mL, the dosage of maltose phosphorylase is 0.1 - 10000 U / mL, the dosage of β-phosphoglucomutase is 0.1 - 10000 U / mL, the dosage of polyphosphate glucokinase is 0.1 - 10000 U / mL, the dosage of phosphoglucose isomerase is 0.1 - 10000 U / mL, and the dosage of pyrophosphate-dependent phosphofructokinase is 0.1 - 10000 U / mL.

[0061] Preferably, the dosage of starch and / or starch derivatives is 20 g / L, the dosage of polyphosphates is 20 mM, the dosage of pyrophosphates is 100 mM, the dosage of β-amylase is 2 U / mL, the dosage of maltose phosphorylase is 2 U / mL, the dosage of β-phosphoglucomutase is 2 U / mL, the dosage of polyphosphate glucokinase is 2 U / mL, the dosage of phosphoglucose isomerase is 2 U / mL, and the dosage of pyrophosphate-dependent phosphofructokinase is 2 U / mL.

[0062] The enzymatic catalysis reaction system is carried out at 30 - 90 °C, and the reaction time is 1 - 200 hours. Preferably, the reaction temperature is 50 °C and the reaction time is 72 hours.

[0063] In the above-mentioned multi-enzyme reaction system, a buffer solution, inorganic phosphorus, and magnesium salt are also included. The concentration of inorganic phosphorus is 5 - 500 mM; more preferably, the concentration of inorganic phosphorus is 10 mM. The buffer solution is preferably a phosphate buffer solution, the pH of the buffer solution is 4.0 - 10.0, and the concentration of the buffer solution is 5 - 500 mM; more preferably, the concentration of the buffer solution is 10 mM and the pH is 6.5 - 7.0. The magnesium salt is magnesium sulfate, and the concentration is 0.01 - 500 mM; more preferably, the concentration of magnesium sulfate is 25 mM.

[0064] According to an embodiment of the present invention, the wheat β-amylase (β-amylase, EC 3.2.1.2) of the present invention can hydrolyze α-1,4 glycosidic bonds, act on the non-reducing end of the substrate, and generate maltose. The optimal pH range of the β-amylase is 6.0 - 8.0, which can adapt to the reaction conditions of the multi-enzyme reaction system. The β-amylase can also be a mutant enzyme with the same function obtained by protein engineering modification. For example, the β-amylase is derived from Bacillus cereus BQ10-S1 SpoII.

[0065] The maltose phosphorylase (maltose phosphorylase, EC 2.4.1.8) of the present invention can be selected from any enzyme having the function of converting maltose into D-glucose and β-D-glucose 1-phosphate, including mutant enzymes with the same function obtained by protein engineering modification. For example, the sources of the maltose phosphorylase include, but are not limited to, Bacillus sp. RK-1, Lactobacillus acidophilus, Paenibacillus sp. SH-55, Lactobacillus brevis, Bacillus subtilis, etc. Preferably, it is the maltose phosphorylase derived from Bacillus sp. RK-1.

[0066] The β-phosphoglucomutase (β-phosphoglucomutase, EC 5.4.2.6) of the present invention can be selected from any enzyme having the function of converting β-D-glucose 1-phosphate into β-D-glucose 6-phosphate, including mutant enzymes with the same function obtained by protein engineering modification. For example, the sources of the β-phosphoglucomutase include, but are not limited to, Pyrococcus sp. strain ST04, Pyrococcus horikoshii OT3, Thermococcus barophilus, etc. Preferably, it is the β-phosphoglucomutase derived from Pyrococcus horikoshii OT3.

[0067] The polyphosphate glucokinase (EC 2.7.1.63) described in the present invention can be selected from any enzyme having the function of converting glucose and polyphosphate into α-D-glucose 6-phosphate, including mutant enzymes obtained by protein engineering with equivalent functions. For example, the sources of the polyphosphate glucokinase include, but are not limited to, Thermobifida fusca, Corynebacterium glutamicum, Propionibacterium shermanii, etc. Preferably, it is the polyphosphate glucokinase derived from Thermobifida fusca.

[0068] The phosphoglucose isomerase (EC 5.3.1.9) described in the present invention can be selected from any enzyme having the function of converting glucose 6-phosphate into fructose 6-phosphate, including mutant enzymes obtained by protein engineering with equivalent functions. The sources of the phosphoglucose isomerase include, but are not limited to, Thermus thermophilus, Pyrococcus furiosus, Clostridium thermocellum, etc. Preferably, it is the phosphoglucose isomerase derived from Thermus thermophilus.

[0069] The inositol-1-phosphate synthase (EC 5.5.1.4) described in the present invention can be selected from any enzyme having the function of converting glucose 6-phosphate into inositol 1-phosphate, including mutant enzymes obtained by protein engineering with equivalent functions. The sources of the inositol-1-phosphate synthase include, but are not limited to, Thermococcus kodakarensis, Archaeoglobus fulgidus, Saccharomyces cerevisiae, etc. Preferably, it is the inositol-1-phosphate synthase derived from Archaeoglobus fulgidus.

[0070] The inositol monophosphatase (Inositol monophosphatase, EC 3.1.3.25) described in the present invention can be selected from any enzyme having the function of dephosphorylating inositol 1-phosphate to generate inositol, including mutant enzymes with equivalent functions obtained by protein engineering modification. The sources of the inositol monophosphatase include, but are not limited to, Thermotoga maritima MSB8, Escherichia coli, Mycobacterium smegmatis, etc. Preferably, it is the inositol monophosphatase derived from Thermotoga maritima MSB8.

[0071] The tagatose 6-phosphate 4-epimerase (tagatose 6-phosphate 4-epimerase, EC 5.1.3.40) described in the present invention can be replaced by any enzyme having the function of converting fructose 6-phosphate to tagatose 6-phosphate, including mutant enzymes with equivalent functions obtained by protein engineering modification. The sources of the tagatose 6-phosphate 4-epimerase include, but are not limited to, Agrobacterium tumefaciens C58, Dictyoglomus thermophilum, Thermoanaerobacter indiensis, etc. Preferably, it is the tagatose 6-phosphate 4-epimerase derived from Thermoanaerobacter indiensis.

[0072] The D-allulose 6-phosphate 3-epimerase (D-allulose 6-phosphate 3-epimerase, EC 5.1.3.-) described in the present invention can be selected from any enzyme having the function of converting fructose 6-phosphate to D-allulose 6-phosphate, including mutant enzymes with equivalent functions obtained by protein engineering modification. The sources of the D-allulose 6-phosphate 3-epimerase include, but are not limited to, Escherichia coli, Thermoanaerobacterium thermosaccharolyticum DSM 571, etc. Preferably, it is the D-allulose 6-phosphate 3-epimerase derived from Thermoanaerobacterium thermosaccharolyticum DSM 571.

[0073] The mannose 6-phosphate isomerase (EC 5.3.1.8) described in the present invention may be selected from any enzyme having the function of converting fructose 6-phosphate into mannose 6-phosphate, including mutant enzymes with equivalent functions obtained by protein engineering. The sources of the mannose 6-phosphate isomerase include but are not limited to Thermus thermophilus, Thermotoga maritima MSB8, Archaeoglobus fulgidus, etc. Preferably, it is the mannose 6-phosphate isomerase derived from Thermus thermophilus.

[0074] The phosphoglucose / phosphomannose isomerases (EC 5.3.1.9, EC 5.3.1.8) described in the present invention may be replaced by any enzyme having the function of converting glucose 6-phosphate into mannose 6-phosphate, including mutant enzymes with equivalent functions obtained by protein engineering. The sources of the phosphoglucose / phosphomannose isomerases include but are not limited to Aeropyrum pernix, Thermoplasma acidophilum, Dictyoglomus thermophilum, etc. Preferably, it is the phosphoglucose / phosphomannose isomerases derived from Dictyoglomus thermophilum.

[0075] The pyrophosphate-dependent phosphofructokinase (EC 2.7.1.90) described in the present invention may be replaced by any enzyme having the function of generating fructose 1,6-diphosphate and inorganic phosphorus from fructose 6-phosphate and pyrophosphate, or by a mutant enzyme with equivalent functions obtained by protein engineering. The sources of the pyrophosphate-dependent phosphofructokinase include but are not limited to Thermotoga maritima, Dictyoglomus thermophilum, Thermoproteus tenax, etc. Preferably, it is the 6-phosphofructokinase derived from Thermotoga maritima.

[0076] The phosphatase described in the present invention may be phytase, acid phosphatase, alkaline phosphatase, and specific phosphatase. Phosphatases with good substrate specificity are preferred, such as one of tagatose 6-phosphate specific phosphatase, psicose 6-phosphate specific phosphatase, and mannose 6-phosphate specific phosphatase. When psicose 6-phosphate is used as the substrate, psicose 6-phosphate phosphatases derived from Acidothermus cellulolyticus, Escherichia coli, Clostridium thermocellum, and Bacteroides fragilis NCTC 9343 are preferred, and their protein numbers correspond one by one to Acel_0099 (KEGG), b3399 (KEGG), Cthe_0261 (KEGG), and BF9343_0892. More preferably, it is the psicose 6-phosphate phosphatase corresponding to the protein number Cthe_0261 (KEGG). When mannose 6-phosphate is used as the substrate, phosphatases derived from Thermotoga maritima MSB8, Pseudomonas putida, and Pseudomonas syringae are preferred, and their protein numbers correspond one by one to TM0651 (KEGG), PP_1764 (KEGG), and PSPPH_2719. More preferably, it is the mannose 6-phosphate phosphatase corresponding to the protein number TM0651 (KEGG). When tagatose 6-phosphate is used as the substrate, the tagatose 6-phosphate phosphatase derived from Archaeoglobus fulgidus, with the protein number AF_0444 (KEGG), is preferred.

[0077] The present invention provides a method for utilizing starch and / or starch derivatives, mainly by degrading starch to maltose using β-amylase (β-amylase, EC 3.2.1.2); subsequently, maltose generates equimolar amounts of β-D-glucose 1-phosphate and glucose under the action of maltose phosphorylase (maltose phosphorylase, EC 2.4.1.8), β-D-glucose 1-phosphate generates β-D-glucose 6-phosphate under the action of β-phosphoglucomutase (β-phosphoglucomutase, EC 5.4.2.6), and glucose generates α-D-glucose 6-phosphate under the action of polyphosphate glucokinase (polyphosphate glucokinase, EC 2.7.1.63). The method of the present invention has the following advantages: (1) The traditional process for preparing maltose from starch is carried out under acidic conditions (pH 5.0 - 5.5). By developing a β-amylase that can maintain high activity under neutral conditions, the optimal reaction conditions of various enzymes in the whole reaction are matched with each other, and the whole reaction can be carried out in a one-pot method, directly preparing high-value-added products from starch or its derivatives in one step, simplifying the operation steps and saving costs. (2) Since the method of the present invention uses a one-pot method, there is no need for intermediate separation steps, the reaction yield is high, and there is no need to add other enzymes to promote starch utilization in the later stage. At the same time, the present invention also provides routes for preparing various products such as inositol, tagatose, allulose, mannose, and fructose 1,6-diphosphate. Generally speaking, the present invention has high starch utilization efficiency, is easy to operate, realizes the preparation of diversified products and a high product yield, and improves the utilization value of starch and starch derivatives. BRIEF DESCRIPTION OF THE DRAWINGS

[0078] Figure 1 It is a catalytic pathway diagram for preparing glucose 6-phosphate from starch by enzymatic method; MA, β-amylase, β-amylase; MP, maltose phosphorylase, maltose phosphorylase; β-PGM, β-phosphoglucomutase, β-glucose phosphomutase; PPGK, polyphosphate glucokinase, polyphosphate-dependent glucokinase.

[0079] Figure 2 It is a catalytic pathway diagram for preparing inositol from starch by enzymatic method; IPS, Inositol 1-phophate synthase, inositol 1-phosphate synthase; IMP, inositol monophosphatase, inositol monophosphatase.

[0080] Figure 3It is a catalytic pathway diagram for preparing tagatose from starch by enzymatic catalysis; PGI, phosphoglucose isomerase; T6PE, tagatose 6-phosphate 4-epimerase; T6PP, tagatose6-phosphate phosphatase.

[0081] Figure 4 It is a catalytic pathway diagram for preparing allulose from starch by enzymatic catalysis; A6PE, D-allulose 6-phosphate3-epimerase; A6PP, allulose 6-phosphatephosphatase.

[0082] Figure 5 It is a catalytic pathway diagram for preparing mannose from starch by enzymatic catalysis; MPI, mannose 6-isomerase; M6PP, mannose 6-phosphate phosphatase.

[0083] Figure 6 It is a catalytic pathway diagram for preparing fructose 1,6-diphosphate from starch by enzymatic catalysis; PPi-PFK, 6-phosphofructokinase. Detailed implementation manners

[0084] The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer as the description progresses. However, it should be understood that the described embodiments are exemplary only and do not constitute any limitation to the scope of the present invention. Those skilled in the art should understand that the details and forms of the technical solutions of the present invention can be modified or replaced without departing from the spirit and scope of the present invention, but such modifications or replacements all fall within the protection scope of the present invention.

[0085] Example 1: Preparation of glucose 6-phosphate from maltodextrin

[0086] Convert maltodextrin and polyphosphate into glucose 6-phosphate through an in vitro multi-enzyme catalytic system, and the reaction pathway is as Figure 1As shown, the key enzymes involved in this pathway are: (1) β-amylase (β-amylase, EC 3.2.1.2), which can start from the non-reducing end of starch and hydrolyze α-1,4 glycosidic bonds successively in units of maltose; (2) maltose phosphorylase (maltosephosphorylase, EC 2.4.1.8), which catalyzes the phosphorolysis of maltose to produce D-glucose and β-D-glucose 1-phosphate; (3) β-phosphoglucomutase (β-phosphoglucomutase, EC 5.4.2.6), which catalyzes the conversion of β-D-glucose 1-phosphate to β-D-glucose 6-phosphate; (4) polyphosphate glucokinase (polyphosphate glucokinase, EC 2.7.1.63), which catalyzes the formation of D-glucose 6-phosphate from D-glucose and polyphosphate.

[0087] In this example, β-amylase is derived from Bacillus cereus BQ10-S1 Spo II, and its protein number is P36924 (Uniprot); maltose phosphorylase is derived from Bacillus sp. RK-1, and its protein numbers are AB084460.1 (Genebank); β-phosphoglucomutase is derived from Pyrococcus horikoshii, and its protein number is PH0749 (KEGG); polyphosphate glucokinase is derived from Thermobifida fusca YX, and its protein number is Tfu_1811. These genes are obtained by gene synthesis, cloned onto the pET vector, and the corresponding expression plasmids pET20b-BcMA, pET20b-BsMP, pET20b-PhβPGM, and pET28a-TfuPPGK are obtained respectively. Subsequently, they are transferred into Escherichia coli BL21(DE3) for protein expression and purification to obtain the corresponding enzymes.

[0088] In a 1.0 mL reaction system, it contains 20 g / L maltodextrin (DE 4-7), 20 mM sodium phosphate buffer (pH 7.0), 100 mM HEPES buffer (pH 7.0), 20 mM magnesium sulfate, 20 mM sodium polyphosphate, 2 U / mL β-amylase, 2 U / mL maltose phosphorylase, 2 U / mL β-phosphoglucomutase, and 2 U / mL polyphosphate glucokinase. The catalytic reaction is carried out at 50 °C for 24 hours. The concentration of glucose 6-phosphate is measured using a glucose 6-phosphate assay kit (Sigma-Aldrich, catalog number MAK014).

[0089] Experimental results: After 24 hours of reaction, the concentration of glucose 6-phosphate reached 18.5 g / L, and the conversion rate of glucose 6-phosphate to starch (relative to the substrate maltodextrin) was 92.5%.

[0090] Example 2: Preparation of inositol using starch and polyphosphate

[0091] The catalytic pathway for converting starch and polyphosphate into inositol through an in vitro multi-enzyme catalytic system is shown in Figure 2 . The key enzymes involved in this pathway are: (1) β-amylase (β-amylase, EC 3.2.1.2), which can start from the non-reducing end of starch and hydrolyze α-1,4 glycosides successively in units of maltose; (2) maltose phosphorylase (maltose phosphorylase, EC 2.4.1.8), which catalyzes the phosphorolysis of maltose to generate D-glucose and β-D-glucose 1-phosphate; (3) β-phosphoglucomutase (β-phosphoglucomutase, EC 5.4.2.6), which catalyzes the conversion of β-D-glucose 1-phosphate to β-D-glucose 6-phosphate; (4) polyphosphate glucokinase (polyphosphate glucokinase, EC 2.7.1.63), which catalyzes the formation of D-glucose 6-phosphate from D-glucose and polyphosphate; (5) inositol 1-phosphate synthase (inositol 1-phophate synthase, EC 5.5.1.4), which catalyzes the formation of inositol 1-phosphate from D-glucose 6-phosphate; (6) inositol monophosphatase (inositol monophosphatase, EC 3.1.3.25), which catalyzes the dephosphorylation of inositol 1-phosphate to generate the end product inositol.

[0092] In this example, inositol 1-phosphate synthase is derived from Archaeoglobus fulgidus, and its protein number is AF_1794 (KEGG); inositol monophosphatase is derived from Thermotoga maritima, and its protein number is TM1415 (KEGG). These genes were obtained by gene synthesis and cloned into the pET vector to obtain the corresponding expression plasmids pET20b-AfIPS and pET20b-TmIMP, respectively. Subsequently, they were transferred into Escherichia coli BL21(DE3) for protein expression and purification to obtain inositol 1-phosphate synthase and inositol monophosphatase, respectively. The expression and purification of other enzymes were the same as in Example 1.

[0093] One-pot preparation of inositol using starch as substrate: In a 1.0 mL reaction system, it contains 20 g / L corn starch, 10 mM sodium phosphate buffer (pH 7.0), 100 mM HEPES buffer (pH 7.0), 20 mM magnesium sulfate, 20 mM sodium polyphosphate, 2 U / mL β-amylase, 2 U / mL maltose phosphorylase, 2 U / mL β-phosphoglucomutase, 2 U / mL polyphosphate glucokinase, 2 U / mL inositol 1-phosphate synthase, and 2 U / mL inositol monophosphatase. The catalytic reaction is carried out at 50 °C for 72 hours. The concentrations of inositol and maltose are determined by HPLC. The conditions of HPLC are as follows: HPX-87H chromatographic column (Bio-Rad), 5 mM sulfuric acid solution as the mobile phase, flow rate 0.6 mL / min, column temperature 60 °C, and the detector is a differential refractive index detector.

[0094] Experimental results: After reacting for 72 hours, the concentration of inositol reaches 14.8 g / L, and the conversion rate of inositol to starch is 74.0%.

[0095] Example 3: Preparation of tagatose using maltodextrin and polyphosphate

[0096] The catalytic pathway for converting maltodextrin and polyphosphate into tagatose through an in vitro multi-enzyme catalytic system is shown in Figure 3 . The key enzymes involved in this pathway are: (1) β-amylase (β-amylase, EC 3.2.1.2), which can start from the non-reducing end of starch and hydrolyze α-1,4 glycosidic bonds successively in units of maltose; (2) maltose phosphorylase (maltose phosphorylase, EC 2.4.1.8), which catalyzes the phosphorolysis of maltose to generate D-glucose and β-D-glucose 1-phosphate; (3) β-phosphoglucomutase (β-phosphoglucomutase, EC 5.4.2.6), which catalyzes the conversion of β-D-glucose 1-phosphate to β-D-glucose 6-phosphate; (4) polyphosphate glucokinase (polyphosphate glucokinase, EC 2.7.1.63), which catalyzes the formation of D-glucose 6-phosphate from D-glucose and polyphosphate; (5) phosphoglucose isomerase (phosphoglucose isomerase, EC5.3.1.9), which catalyzes the conversion of glucose 6-phosphate to fructose 6-phosphate; (6) tagatose-6-phosphate epimerase, which catalyzes the epimerization of fructose 6-phosphate to tagatose 6-phosphate; (7) tagatose 6-phosphate phosphatase, which catalyzes the dephosphorylation of tagatose 6-phosphate to generate tagatose.

[0097] In this example, phosphoglucose isomerase is derived from Thermus thermophilus HB8, and its protein number is TTC1710 (KEGG); tagatose 6-phosphate 4-epimerase is derived from Thermoanaerobacter indiensis, and its protein number is WP_019907213.1 (Genebank); tagatose 6-phosphate phosphatase is derived from Archaeoglobus fulgidus, and its protein number is AF_0444 (KEGG). After obtaining these genes by gene synthesis, they were cloned onto the pET vector to obtain the corresponding expression plasmids pET20b-TtcPGI, pET20b-TiT6PE, and pET20b-AfT6PP, respectively. Subsequently, they were transferred into Escherichia coli BL21(DE3) for protein expression and purification to obtain phosphoglucose isomerase, tagatose 6-phosphate 4-epimerase, and tagatose 6-phosphate phosphatase. The expression and purification of other enzymes were the same as in Example 1.

[0098] One-pot preparation of tagatose using maltodextrin as a substrate: A 1.0 mL reaction system contained 20 g / L maltodextrin, 10 mM sodium phosphate buffer (pH 7.0), 100 mM HEPES buffer (pH 7.0), 20 mM magnesium sulfate, 20 mM sodium polyphosphate, 2 U / mL β-amylase, 2 U / mL maltose phosphorylase, 2 U / mL β-glucophosphomutase, 2 U / mL polyphosphoglucose kinase, 2 U / mL phosphoglucose isomerase, 2 U / mL tagatose 6-phosphate epimerase, and 2 U / mL tagatose 6-phosphate phosphatase. The catalytic reaction was carried out at 50 °C for 72 hours. The concentrations of tagatose and maltose were determined by HPLC. The conditions for HPLC were: HPX-87H chromatographic column (Bio-Rad), 5 mM sulfuric acid solution as the mobile phase, flow rate 0.6 mL / min, column temperature 60 °C, and the detector was a differential refractive index detector.

[0099] Experimental results: After reacting for 72 hours, the concentration of tagatose in the reaction system reached 16.5 g / L, and the conversion rate of tagatose relative to maltodextrin was 82.5%.

[0100] Example 4: Preparation of allulose using starch and polyphosphate

[0101] The catalytic pathway for converting starch and polyphosphate into allulose through an in vitro multi-enzyme catalytic system is shown in Figure 4The key enzymes involved in this pathway are: (1) β - amylase (β - amylase, EC 3.2.1.2), which can start from the non - reducing end of starch and hydrolyze α - 1,4 glycosidic bonds successively in units of maltose; (2) maltose phosphorylase (maltose phosphorylase, EC 2.4.1.8), which catalyzes the phosphorolysis of maltose to produce D - glucose and β - D - glucose 1 - phosphate; (3) β - phosphoglucomutase (β - phosphoglucomutase, EC 5.4.2.6), which catalyzes the conversion of β - D - glucose 1 - phosphate to β - D - glucose 6 - phosphate; (4) polyphosphate glucokinase (polyphosphate glucokinase, EC 2.7.1.63), which catalyzes the formation of D - glucose 6 - phosphate from D - glucose and polyphosphate; (5) phosphoglucose isomerase (phosphoglucose isomerase, EC 5.3.1.9), which catalyzes the conversion of glucose 6 - phosphate to fructose 6 - phosphate; (6) D - allulose 6 - phosphate 3 - epimerase (D - allulose 6 - phosphate 3 - epimerase, EC 5.1.3.-), which catalyzes the isomerization of fructose 6 - phosphate to allulose - 6 - phosphate; (7) allulose 6 - phosphate phosphatase, which catalyzes the dephosphorylation of allulose 6 - phosphate to produce allulose.

[0102] In this example, D - allulose 6 - phosphate 3 - epimerase is derived from Thermoanaerobacterium thermosaccharolyticum DSM 571, and its protein number is Tthe_1731 (KEGG); the phosphatase is derived from Clostridium thermocellum, and its protein number is Cthe_0261 (KEGG). After obtaining these genes by gene synthesis, they were cloned into the pET20b vector to obtain the corresponding expression plasmids pET20b - TtA6PE and pET20b - CtA6PP respectively. Subsequently, they were transferred into Escherichia coli BL21(DE3) for protein expression and purification to obtain D - allulose 6 - phosphate 3 - epimerase and allulose 6 - phosphate phosphatase respectively. The expression and purification of other enzymes are the same as those in Example 1 and Example 3.

[0103] One-pot preparation of allulose using starch as a substrate: In a 1.0 mL reaction system, it contains 20 g / L corn starch, 10 mM sodium phosphate buffer (pH 7.0), 100 mM HEPES buffer (pH 7.0), 20 mM magnesium sulfate, 20 mM sodium polyphosphate, 2 U / mL β-amylase, 2 U / mL maltose phosphorylase, 2 U / mL β-phosphoglucomutase, 2 U / mL polyphosphate glucokinase, 2 U / mL phosphoglucose isomerase, 2 U / mL allulose-6-phosphate 3-epimerase, and 2 U / mL allulose-6-phosphate phosphatase. The catalytic reaction is carried out at 50 °C for 72 hours. The concentration of allulose is determined by HPLC. Determination conditions: HPX-87H chromatographic column (Bio-Rad), 5 mM sulfuric acid solution as the mobile phase, flow rate 0.6 mL / min, column temperature 60 °C, and the detector is a differential refractive index detector.

[0104] Experimental results: After reacting for 72 hours, the concentration of allulose in the reaction system reaches 15.0 g / L, and the conversion rate of allulose relative to starch is 75.0%.

[0105] Example 5: Preparation of mannose using starch and polyphosphate

[0106] The catalytic pathway for converting starch and polyphosphate into mannose through an in vitro multi-enzyme catalytic system is shown in Figure 5 . The key enzymes involved in this pathway are: (1) β-amylase (β-amylase, EC 3.2.1.2), which can start from the non-reducing end of starch and hydrolyze α-1,4 glycosidic bonds successively in units of maltose; (2) maltose phosphorylase (maltose phosphorylase, EC 2.4.1.8), which catalyzes the phosphorolysis of maltose to generate D-glucose and β-D-glucose 1-phosphate; (3) β-phosphoglucomutase (β-phosphoglucomutase, EC 5.4.2.6), which catalyzes the conversion of β-D-glucose 1-phosphate into β-D-glucose 6-phosphate; (4) polyphosphate glucokinase (polyphosphate glucokinase, EC 2.7.1.63), which catalyzes the formation of D-glucose 6-phosphate from D-glucose and polyphosphate; (5) phosphoglucose isomerase (phosphoglucose isomerase, EC 5.3.1.9), which catalyzes the conversion of glucose 6-phosphate into fructose 6-phosphate; (6) mannose-6-phosphate isomerase (mannose-6-phosphate isomerase, EC 5.3.1.8), which catalyzes the isomerization of fructose 6-phosphate into mannose 6-phosphate; (7) mannose 6-phosphate phosphatase, which catalyzes the dephosphorylation of mannose 6-phosphate to generate mannose.

[0107] In this example, mannose 6-phosphate isomerase is derived from Thermus thermophilus HB27, and its protein number is TTHA1345 (KEGG); mannose 6-phosphate phosphatase is derived from Thermotoga maritima MSB8, and its protein number is TM0651 (KEGG). After obtaining these genes by gene synthesis, they were cloned onto the PET vector to obtain the corresponding expression plasmids pET20b-TthMPI and pET21a-TmM6PP, respectively. Subsequently, they were transferred into Escherichia coli BL21(DE3) for protein expression and purification, and mannose 6-phosphate isomerase and mannose 6-phosphate phosphatase were obtained respectively. The expression and purification of other enzymes were the same as those in Example 1 and Example 3.

[0108] One-pot preparation of mannose using starch as the substrate: A 1.0 mL reaction system contains 20 g / L corn starch, 10 mM sodium phosphate buffer (pH 7.0), 100 mM HEPES buffer (pH 7.0), 20 mM magnesium sulfate, 20 mM sodium polyphosphate, 2 U / mL β-amylase, 2 U / mL maltose phosphorylase, 2 U / mL β-glucose phosphomutase, 2 U / mL polyphosphoglucokinase, 2 U / mL phosphoglucose isomerase, 2 U / mL mannose 6-phosphate isomerase, and 2 U / mL mannose 6-phosphate phosphatase. The catalytic reaction was carried out at 50 °C for 72 hours. The concentration of mannose was determined by HPLC. Determination conditions: HPX-87H chromatographic column (Bio-Rad), 5 mM sulfuric acid solution as the mobile phase, flow rate 0.6 mL / min, column temperature 60 °C, and the detector was a differential refractive index detector.

[0109] Experimental results: After reacting for 72 hours, the concentration of mannose reached 13.6 g / L, and the conversion rate of mannose relative to starch was 68.0%.

[0110] Example 6: Preparation of fructose 1,6-diphosphate using starch and polyphosphate

[0111] The catalytic pathway for converting starch and polyphosphate into fructose 1,6-diphosphate through an in vitro multi-enzyme catalytic system is shown in Figure 6The key enzymes involved in this pathway are: (1) β - amylase (β - amylase, EC 3.2.1.2), which can hydrolyze α - 1,4 glycosidic bonds sequentially in units of maltose starting from the non - reducing end of starch; (2) maltose phosphorylase (maltosephosphorylase, EC 2.4.1.8), which catalyzes the phosphorolysis of maltose to produce D - glucose and β - D - glucose 1 - phosphate; (3) β - phosphoglucomutase (β - phosphoglucomutase, EC 5.4.2.6), which catalyzes the conversion of β - D - glucose 1 - phosphate to β - D - glucose 6 - phosphate; (4) polyphosphate glucokinase (polyphosphate glucokinase, EC 2.7.1.63), which catalyzes the formation of D - glucose 6 - phosphate from D - glucose and polyphosphate; (5) phosphoglucose isomerase (phosphoglucose isomerase, EC 5.3.1.9), which catalyzes the conversion of glucose 6 - phosphate to fructose 6 - phosphate; (6) pyrophosphate - dependent phosphofructokinase (6 - phosphofructokinase, EC 2.7.1.90), which catalyzes the formation of fructose 1,6 - bisphosphate from pyrophosphate and fructose 6 - phosphate, releasing one molecule of inorganic phosphate at the same time.

[0112] In this example, the pyrophosphate - dependent phosphofructokinase is derived from Thermotoga maritima, with the protein number TM0289 (KEGG). After obtaining this gene by gene synthesis, it was cloned into the PET vector to obtain the corresponding expression plasmid pET20b - TmPPi - PFK. Subsequently, it was transferred into Escherichia coli BL21(DE3) for protein expression and purification. The expression and purification of other enzymes were the same as in Examples 1 and 3.

[0113] One-pot preparation of fructose 1,6-diphosphate using starch as a substrate: In a 1.0 mL reaction system, it contains 20 g / L corn starch, 10 mM sodium phosphate buffer (pH 7.0), 100 mM HEPES buffer (pH 7.0), 25 mM magnesium sulfate, 20 mM sodium polyphosphate, 100 mM sodium pyrophosphate, 10 U / mL β-amylase, 2 U / mL maltose phosphorylase, 2 U / mL β-glucose phosphomutase, 2 U / mL polyphosphoglucose kinase, and 2 U / mL pyrophosphate-dependent phosphofructokinase. The catalytic reaction is carried out at 50 °C for 24 hours. The concentration of fructose 1,6-diphosphate is determined by an enzymatic method (Wang W, Liu M, You C, Li Z, Zhang YP. 2017. ATP-free biosynthesis of a high-energy phosphate metabolite fructose 1,6-diphosphate by in vitro metabolic engineering. Metab Eng 42:168-174.). The detection of maltose is the same as in Example 1.

[0114] Experimental results: After 24 hours of reaction, the concentration of fructose 1,6-diphosphate reached 16.0 g / L, and the conversion rate of fructose 1,6-diphosphate relative to starch was 80.0%.

[0115] As described above, the embodiments of the present invention have been described. However, the present invention is not limited to the above embodiments. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A method for utilizing starch or its derivatives, the method comprising the following reaction steps: (1) using β-amylase to catalyze and convert a substrate into maltose, wherein the substrate is starch and / or a starch derivative; (2) using maltose phosphorylase to catalyze the conversion of maltose and inorganic phosphorus into β-D-glucose 1-phosphate and glucose; (3) using β-glucose phosphomutase to catalyze the conversion of β-D-glucose 1-phosphate into β-D-glucose 6-phosphate; (4) using polyphosphate glucose kinase to catalyze the conversion of glucose and polyphosphate in step (2) into α-D-glucose 6-phosphate; The steps (1)-(4) are carried out by a "one-pot method" to prepare the product.

2. The method according to claim 1, wherein the starch is soluble starch; and / or the polyphosphate is sodium polyphosphate; and / or the inorganic phosphorus is selected from any one of sodium phosphate, sodium dihydrogen phosphate, disodium hydrogen phosphate, potassium phosphate, potassium dihydrogen phosphate and dipotassium hydrogen phosphate.

3. The method according to claim 2, wherein the starch is selected from one or more of corn starch, wheat starch, tapioca starch, and potato starch; and the starch derivative is selected from any one or more of partially hydrolyzed starch, starch dextrin, maltodextrin, or maltopolysaccharide; And / or, the polyphosphate is selected from one of sodium tripolyphosphate and sodium hexametaphosphate or two of them in any ratio.

4. The method according to any one of claims 1 to 3, wherein the concentration of the substrate is 1-800 g / L based on the total volume of the reaction system; the concentration of polyphosphate is 1-500 mM; the amount of β-amylase is 0.1-10000 U / mL; the amount of maltose phosphorylase is 0.1-10000 U / mL; the amount of β-glucose phosphotyrosinase is 0.1-10000 U / mL; the amount of polyphosphate glucokinase is 0.1-10000 U / mL; and the concentration of inorganic phosphorus is 5-500 mM.

5. The method according to claim 4, wherein the concentration of the substrate is 20-200 g / L based on the total volume of the reaction system; and / or, the concentration of polyphosphate is 20-200 mM; and / or, β-amylase is 1-1000 U / mL; and / or, maltose phosphorylase is 1-1000 U / mL; and / or, the dosage of β-glucose phosphomutase is 1-1000 U / mL; and / or, the dosage of polyphosphate glucokinase is 1-1000 U / mL; And / or, the concentration of inorganic phosphorus is 10 mM.

6. The method according to any one of claims 1 to 3, further comprising step (5): α-D-glucose 6-phosphate and β-D-glucose 6-phosphate are further subjected to an enzymatic reaction to obtain a final product, wherein the final product is at least one of inositol, tagatose, psicose, mannose or fructose 1,6-bisphosphate.

7. The method according to claim 6, wherein step (5) comprises any of the following steps: (5a) adding inositol 1-phosphate synthase (EC 5.5.1.4) and inositol monophosphatase (EC 3.1.3.25) to obtain inositol; (5b) adding phosphoglucose isomerase (EC 5.3.1.9), tagatose 6-phosphate 4-epimerase (EC5.1.3.40) and phosphatase to obtain tagatose; (5c) adding phosphoglucose isomerase (EC 5.3.1.9), psicose 6-phosphate 3-epimerase (EC5.1.3.-) and phosphatase to obtain psicose; (5d) adding phosphoglucose isomerase (EC 5.3.1.9), mannose 6-phosphate isomerase (EC 5.3.1.8) and phosphatase to obtain mannose; or (5e) Pyrophosphate is added, along with phosphoglucose isomerase (EC 5.3.1.9) and pyrophosphate-dependent phosphofructokinase (EC 2.7.1.90) to produce fructose 1,6-bisphosphate.

8. The method according to claim 7, wherein steps (1) to (5) are carried out by a "one-pot" reaction.

9. The method according to claim 7 or 8, wherein in step (5), based on the total volume of the reaction system, the amounts of inositol 1-phosphate synthase, inositol monophosphatase, phosphoglucose isomerase, tagatose 6-phosphate 4-epimerase, psicose 6-phosphate 3-epimerase, mannose 6-phosphate isomerase, pyrophosphate-dependent phosphofructokinase and phosphatase are 0.1-10000 U / mL respectively; And / or, the phosphatase is any one of phytase, acid phosphatase, alkaline phosphatase or specific phosphatase.

10. The method according to claim 9, wherein in step (5), based on the total volume of the reaction system, the amounts of inositol 1-phosphate synthase, inositol monophosphatase, phosphoglucose isomerase, tagatose 6-phosphate 4-epimerase, psicose 6-phosphate 3-epimerase, mannose 6-phosphate isomerase, pyrophosphate-dependent phosphofructokinase and phosphatase are 1-1000 U / mL, respectively; And / or, the phosphatase is at least one of tagatose 6-phosphate specific phosphatase, psicose 6-phosphate specific phosphatase or mannose 6-phosphate specific phosphatase.

11. The method according to claim 10, wherein in the step (5), based on the total volume of the reaction system, the amounts of inositol 1-phosphate synthase, inositol monophosphatase, phosphoglucose isomerase, tagatose 6-phosphate 4-epimerase, psicose 6-phosphate 3-epimerase, mannose 6-phosphate isomerase, pyrophosphate-dependent phosphofructokinase and phosphatase are 2-100 U / mL, respectively.

12. The method according to any one of claims 1 to 3, wherein the reaction system further comprises any one or more of a buffer and / or a magnesium salt.

13. The method according to claim 12, wherein the buffer is selected from any one or more of HEPES buffer, Tris-HCl buffer, MOPS buffer, citrate buffer, and phosphate buffer; And / or, the magnesium salt is selected from magnesium chloride or magnesium sulfate.

14. The method according to claim 13, wherein in the reaction system, the pH of the buffer solution is 4.0-10.0, and the concentration of the buffer solution is 5-500 mM; And / or, the magnesium salt concentration is 0.01-500 mM.

15. The method according to any one of claims 1 to 3, wherein a starch debranching enzyme is further added to the reaction system, or the starch or starch derivative is pretreated with a starch debranching enzyme before step (1).

16. The method according to claim 15, wherein the starch debranching enzyme is any one or both of isoamylase (EC 3.2.1.68) or pullulanase (EC 3.2.1.41); And / or, the dosage of the starch debranching enzyme is based on the total volume of the reaction system, and the concentration of the debranching enzyme is 0.1-10000 U / mL.

17. The method according to claim 16, wherein the starch debranching enzyme is used in an amount based on the total volume of the reaction system, and the concentration of the debranching enzyme is 1-1000 U / mL.

18. The method according to claim 17, wherein the starch debranching enzyme is used in an amount based on the total volume of the reaction system, and the concentration of the debranching enzyme is 2-100 U / mL.

19. The method according to any one of claims 1 to 3, wherein the reaction is carried out at a temperature of 30-90°C and a reaction time of 1-200 hours.

Images