CN114990170A - Method for co-producing fructose and gluconic acid or gluconate by enzyme catalysis of glucose - Google Patents

Method for co-producing fructose and gluconic acid or gluconate by enzyme catalysis of glucose Download PDF

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CN114990170A
CN114990170A CN202210777010.0A CN202210777010A CN114990170A CN 114990170 A CN114990170 A CN 114990170A CN 202210777010 A CN202210777010 A CN 202210777010A CN 114990170 A CN114990170 A CN 114990170A
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fructose
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林建强
李�灿
林慧彬
林建群
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Qingdao Longding Biotechnology Co ltd
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Abstract

The invention discloses a method for catalyzing glucose to co-produce fructose and gluconic acid or gluconate. The method comprises the steps of taking D-glucose as a raw material, firstly adding glucose isomerase to convert the D-glucose into D-fructose, removing the glucose isomerase after reaction, and stopping the mutual conversion reaction between the D-glucose and the D-fructose; then, glucose oxidase and catalase were added to the reaction solution to convert the unconverted D-glucose into gluconic acid. The generated gluconic acid is separated from the D-fructose by using anion exchange resin, and the D-fructose and the gluconic acid with high purity are respectively obtained. Gluconic acid can react with the metal alkali solution to generate corresponding gluconate. The invention obtains high-purity D-fructose and gluconic acid or gluconate by utilizing enzyme catalysis and a simple separation method, has simple process and high efficiency, can reduce the production cost of the D-fructose, increases the product variety and has wide industrial application prospect.

Description

Method for co-producing fructose and gluconic acid or gluconate by enzyme catalysis of glucose
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a method for enzyme-catalyzed glucose co-production of fructose and gluconic acid or gluconate.
Background
D-fructose is the most sweet natural sweetener, has the sweetness about 3 times that of D-glucose and 1.2-1.8 times that of cane sugar, and has pure sweetness and pure mouthfeel, and does not obscure the flavors of other substances in the food. The D-fructose metabolism is independent of insulin, and cannot cause excessive fluctuation of blood sugar. Due to the excellent characteristics, D-fructose is widely used in the food and pharmaceutical industries.
D-fructose can be produced by D-glucosidase isomerization, the substrate conversion rate is 45-55%, and syrup containing D-fructose is industrially produced in large quantities and used. In order to improve the content of the D-fructose, a chromatographic separation technology is needed to realize the separation of the D-fructose and the D-glucose, so that a high-purity product with the content of the D-fructose of more than 95 percent can be produced, and then the D-fructose is crystallized to prepare the crystallized D-fructose with the purity of more than 98 percent. However, due to the consistent molecular weight of D-fructose and D-glucose, other physicochemical properties are also very close, resulting in difficulty in separating D-fructose from D-glucose. At present, the simulated moving bed chromatography technology is generally adopted to separate D-fructose and D-glucose, but the equipment is complex and expensive, and the high-efficiency, low-cost and large-scale production of high-purity D-fructose is not facilitated. The high-purity D-fructose is high in price due to the reasons, so that the market application of the high-purity D-fructose is hindered. Therefore, there is still a need for a process for producing high purity D-fructose with high efficiency and low cost.
Gluconic acid is an important sour agent and has important application in the industries of food, beverage and the like. Some gluconates, such as potassium gluconate, sodium gluconate, calcium gluconate, iron gluconate, zinc gluconate, magnesium gluconate and the like can be used as essential metal element supplements for human bodies and used as nutriments to supplement trace elements required by the human bodies, and have high commercial value. Therefore, the residual D-glucose in the production process of the D-fructose is converted into gluconic acid or gluconate which is easy to separate from the D-fructose, so that the separation and purification process of the D-fructose is simplified, the separation and purification cost is reduced, the product variety is increased, and the economic benefit is improved.
The gluconic acid production method mainly comprises a chemical catalysis method, an electrolytic oxidation method, a microbial fermentation method and an enzyme catalysis method. The enzyme catalysis method has the characteristics of being natural and green, strong in reaction specificity, high in product purity, high in concentration, high in conversion efficiency and the like, and is increasingly the mainstream process. However, no relevant report about the co-production of D-fructose and gluconic acid or gluconate by an enzyme catalysis method exists at present.
Disclosure of Invention
The invention aims to provide a method for co-producing fructose and gluconic acid or gluconate by enzyme catalysis of glucose, which has the advantages of simple and effective process, low cost and high yield.
In order to realize the purpose of the invention, the invention adopts the following technical scheme to realize:
the invention provides a method for co-producing fructose and gluconic acid or gluconate by catalyzing glucose through enzyme, which comprises the following steps:
(1) preparing a D-glucose solution by using purified water by taking D-glucose as a raw material;
(2) adding the D-glucose solution obtained in the step (1) into glucose isomerase for catalytic reaction to obtain a reaction solution, centrifuging, and taking a supernatant;
(3) adding glucose oxidase and catalase into the supernatant obtained in the step (2), carrying out catalytic reaction to obtain a reaction solution, centrifuging, and taking the supernatant;
(4) enabling the supernatant fluid obtained in the step (3) to flow through an adsorption resin column to adsorb gluconic acid, and filtering to remove the adsorption resin to obtain a high-purity D-fructose permeate;
(5) desorbing the adsorption resin removed by filtering in the step (4) by using an acid solution to obtain a gluconic acid solution with higher purity;
(6) and (4) adding the gluconic acid solution obtained in the step (5) into an alkali solution containing metal ions until the pH is neutral to obtain a gluconic acid salt solution containing the metal ions.
Further, the concentration of the D-glucose solution in the step (1) is 10% -80%, and the D-glucose solution is prepared by adding purified water into D-glucose crystals.
Preferably, the concentration of the D-glucose solution in the step (1) is 60%.
Further, in the step (2), free or immobilized glucose isomerase is selected for glucose isomerization, and the dosage of the free or immobilized glucose isomerase is 1-30% of the mass of the D-glucose solution. In the invention, the immobilized enzyme is convenient to take out and can be used repeatedly without heating to inactivate the enzyme; free enzyme needs to be inactivated at high temperature and removed centrifugally, can be used only once, and the color of sugar liquor is easy to brown in the heating process.
Further, the conditions of the catalytic reaction in the step (2) are as follows: the catalysis pH is 7.0-9.0, the catalysis temperature is 65-75 ℃, the catalysis speed is 90-110 rpm, and the catalysis time is 4-10 h.
Preferably, the conditions for the catalytic reaction in the step (2) are as follows: the catalysis pH is 8.0, the catalysis temperature is 70 ℃, the catalysis speed is 100 rpm, and the catalysis time is 8 h.
Further, in the step (3), the glucose oxidase is selected from free or immobilized glucose oxidase, and the dosage of the glucose oxidase is 1-30% of the mass of the D-glucose solution; the catalase is selected from free or immobilized catalase, and the dosage of the catalase is 1-30% of the mass of the D-glucose solution.
Preferably, the dosage of the glucose oxidase and the catalase in the step (3) is respectively 10% and 6% of the mass of the D-glucose solution.
Further, the conditions of the catalytic reaction in the step (3) are as follows: the catalysis pH is 4-5, the catalysis temperature is 50-60 ℃, the catalysis speed is 90-110 rpm, and the catalysis time is 3-8 h.
Preferably, the conditions for the catalytic reaction in the step (3) are as follows: the catalysis pH is 4.5, the catalysis temperature is 55 ℃, the catalysis speed is 100 rpm, and the catalysis time is 8 h.
Further, the adsorption resin column in the step (4) is an anion exchange resin column, specifically a macroporous styrene series weak-base anion exchange resin column; the purity of the D-fructose in the permeate is not lower than 95%.
Further, the acid solution in the step (5) is dilute hydrochloric acid, and the concentration of the dilute hydrochloric acid is 0.5M.
Further, the metal ions in the step (6) comprise potassium, sodium, calcium, iron, zinc and magnesium; the concentration of the alkali solution was 5M or a saturated solution at room temperature.
Further, in the step (2) or (3), if immobilized glucose isomerase, glucose oxidase or catalase is selected, the reaction solution does not need to be heated to inactivate the enzyme; if free glucose isomerase, glucose oxidase or catalase is selected, heating and enzyme deactivation treatment needs to be carried out on the reaction liquid, and the steps are as follows: heating the reaction solution to 100 ℃ and maintaining for 20min to inactivate the enzyme; cooling to room temperature, centrifuging at 8000-10000 rpm for 10-20 min, and collecting supernatant.
Further, in the enzyme inactivation in the step (2) or (3), the free enzyme is heated to denature and inactivate and is centrifuged at 10000 rpm for 20min to remove denatured enzyme protein; the immobilized enzyme was centrifuged at 8000 rpm for 20min to remove the solid catalyst.
Compared with the prior art, the invention has the following advantages and beneficial effects:
the method utilizes two-step enzyme catalytic reaction to obtain D-fructose and gluconic acid, and utilizes excessive ion exchange resin to adsorb and remove the gluconic acid to obtain D-fructose solution with higher content and higher purity; the gluconic acid solution obtained after the resin desorption can be conveniently converted into the gluconic acid salt solution, and the product value is high. The method provided by the invention overcomes the difficult problem of chromatographic separation of D-fructose and D-glucose by converting D-glucose into gluconic acid/gluconate, does not need complex separation equipment, has simple process and low cost, is easy to realize industrial production, increases the product variety and product value, and has huge application prospect.
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FIG. 1 is a schematic diagram of the enzymatic method of the present invention, in which M is a 1-valent alkali metal (which may be a divalent or higher-valent metal).
FIG. 2 shows a D-fructose HPLC chromatogram and a standard curve.
FIG. 3 shows the effects of (A) pH (D-fructose as substrate), (B) (D-glucose as substrate) and (C) temperature on the enzyme activity of immobilized GI (D-fructose as substrate); (D) influence of temperature on the thermostability of immobilized GI (D-fructose as substrate).
FIG. 4 shows the effect of different resins on the immobilization of glucose oxidase (-G stands for glutaraldehyde cross-linking).
FIG. 5 shows the effect of different amounts of resin (A), glutaraldehyde concentration (B), time (C) and immobilization temperature (D) on the immobilization of glucose oxidase.
Detailed Description
The technical solution of the present invention will be further described in detail with reference to the following specific examples. In the following examples, unless otherwise specified, the experimental methods used were all conventional methods, and materials, reagents and the like used were all available from biological or chemical reagents companies.
The method comprises the steps of converting D-glucose into D-fructose by using glucose isomerase, balancing the reaction to obtain a mixed solution of the D-glucose and the D-fructose (the two are about 50 percent respectively), deactivating the enzyme to terminate the isomerization reaction, adding glucose oxidase and catalase, oxidizing the glucose by the glucose oxidase to generate gluconic acid and hydrogen peroxide which has an inhibition effect on the glucose oxidase, and performing enzymolysis on the hydrogen peroxide by the catalase to generate oxygen and water to remove the inhibition effect of the hydrogen peroxide on the glucose oxidase and promote the glucose oxidation reaction (figure 1). The gluconic acid generated by the invention has good market value, and can be converted into various gluconates to increase product types and further improve the market value. The invention simplifies the production process, increases the product types and the product value, and has good application prospect.
The glucose isomerase, glucose oxidase and catalase in the present invention are all commercially available products. The immobilized glucose oxidase and catalase were prepared by the following procedures. The immobilized enzyme can be used repeatedly, so that the cost of the enzyme is saved; in addition, the immobilized enzyme is easy to separate from the conversion liquid, the step of heating for enzyme deactivation is not needed, energy is saved, and the browning of the sugar liquid caused by heating is avoided.
Example 1 measurement of glucose isomerase Activity
(1) Glucose isomerase activity determination: 100 mmol/L D-glucose or D-fructose solution (pH value 8.0) is used as a substrate, 100 mg of free enzyme powder or immobilized enzyme is added into each milliliter of the substrate solution, the reaction is carried out for 10 min at 70 ℃, the enzyme is immediately boiled in boiling water for 20min to inactivate the enzyme, the reaction solution is 10000 rpm, the temperature is room temperature, the centrifugation is carried out for 10 min, a PVDF 0.45 mu m filter is used for filtering, and the generation or consumption of the D-fructose is measured by HPLC.
(2) D-fructose HPLC analytical method: 100 mg and 10 mg of D-fructose standard are weighed respectively, are fully dissolved by 1 mL of double distilled water to be used as mother liquor, and then are diluted to different concentrations by the double distilled water respectively. The dilution concentrations (g/L) of D-fructose were 20, 40, 60, 80, and 100, respectively. The prepared standards with different concentrations were centrifuged at 10000 rpm for 10 min, filtered through a PVDF 0.45 μm filter, and then measured by HPLC.
HPLC detection conditions: chromatographic column Carbomix Pb-NP column (10: 8%, 7.8X 300 mm, 10 μm, Seiki technology); a difference detector; mobile phase: ddH 2 O, the flow rate is 0.5 mL/min; the sample volume is 10 mu L; the column temperature was 78 ℃.
As a result, referring to FIG. 2, the retention time of D-fructose was about 24 min. The concentrations of D-fructose were plotted on the abscissa and the peak areas on the ordinate to prepare standard curves (FIG. 2). The standard curve equation of the D-fructose is as follows: y is F =3.4×10 5 X F +15077（R 2 = 0.99995), the concentration (g/L) of D-psicose was calculated from the peak area thereof.
Example 2 optimum reaction conditions for immobilized glucose isomerase
(1) Optimum pH: preparing 100 mmol/L D-glucose or D-fructose solution with pH of 2-pH 9, adding 100 mg of commercial immobilized glucose isomerase, reacting at 70 deg.C for 10 min, separating immobilized enzyme from reaction solution, filtering with a PVDF 0.45 μm filter at room temperature at 10000 rpm, centrifuging for 10 min, and measuring D-fructose generation or consumption by HPLC. As shown in FIGS. 3A-3B, the optimum pH of the immobilized glucose isomerase was 8.0.
(2) Optimum temperature and thermal stability: the enzyme activities of the immobilized glucose isomerases were measured at 20 to 90 ℃ respectively. As a result, as shown in FIG. 3C, the optimum temperature for immobilizing glucose isomerase was 70 ℃.
And (3) storing the immobilized glucose isomerase at 20-70 ℃ for different times, and measuring the enzyme activity of the immobilized enzyme. As a result, as shown in FIG. 3D, the enzyme activity of the immobilized glucose isomerase was maintained at 80% or more after 6 hours of storage at 70 ℃.
Example 3 immobilization and optimization of glucose oxidase
1. Glucose oxidase activity assay
The enzyme activity determination method of the glucose oxidase comprises the following steps: 100 mmol/L D-glucose solution (pH value 5.0) with saturated air is used as substrate, 100 mg enzyme is added into each milliliter of substrate solution, after reaction at 50 ℃ for 2 min, the substrate solution is immediately boiled in boiling water for 10 min to inactivate the enzyme, reaction liquid is 10000 rpm, the room temperature is kept, the centrifugation is carried out for 10 min, PVDF 0.45 mu m filter is used for filtration, and the generation amount of the gluconic acid is measured by HPLC.
HPLC detection conditions: chromatography column Aminex HPX-87H column (300X 7.8mm, 9 μm, BIO-RAD, USA); a difference detector; mobile phase: 0.5 mmol/L sulfuric acid, the flow rate is 0.4 mL/min; the sample volume is 10 mu L; the column temperature was 55 ℃.
Definition of enzyme activity: under the condition of the optimal enzyme activity (pH value of 5.0 and 50 ℃), the amount of the enzyme required for catalyzing 1 mg of D-glucose to be converted into gluconic acid per minute is one enzyme activity unit.
Definition of relative enzyme activity: the highest enzyme activity is defined to be 100%, and the enzyme activities of other conditions account for the percentage of the highest enzyme activity.
2. Immobilization and optimization of glucose oxidase
(1) Activation and crosslinking method of immobilized carrier amino resin
Soaking appropriate amount of amino resin in 100 mM K 2 HPO 4 -KH 2 PO 4 (pH 5.0) in buffer, at 25 deg.C, 120 rpm, activated for 5 h.
After the activation, adding a proper amount of glutaraldehyde solution into the buffer solution, crosslinking for 5 h at 25 ℃ and 120 rpm, and then using K 2 HPO 4 -KH 2 PO 4 The glutaraldehyde cross-linked resin was washed with buffer 3 times to wash away residual glutaraldehyde solution to obtain a modified resin.
(2) Comparison of different resins and immobilization methods
Amino resin EA, ion exchange resins SQ, D293, D201, D202 and H2888 are respectively selected as immobilized carriers to carry out enzyme immobilization research. Weighing 1 g of activated resin with different types, wherein each resin is divided into two experimental groups of glutaraldehyde crosslinking and non-crosslinking; mixing the resin and the glucose oxidase liquid according to the proportion that 1 mL of liquid enzyme is added into every 1 g of resin, and immobilizing for 10 hours at 25 ℃ and 120 rpm; by K 2 HPO 4 -KH 2 PO 4 Washing the prepared immobilized enzyme for 3 times by using a buffer solution to completely remove free enzyme liquid to obtain immobilized glucose oxidase; and measuring relative enzyme activity, and comparing the immobilization effects of different resins.
As a result, as shown in FIG. 4, amino resin EA was crosslinked with glutaraldehyde and then immobilized with glucose oxidase at the best effect. The principle is that after glutaraldehyde crosslinking, aldehyde groups on a carrier react with amino groups on enzyme molecules to form Schiff base for enzyme immobilization.
(3) Optimization of resin dosage, glutaraldehyde concentration, immobilization time and immobilization temperature
Different ratios of the resin dosage to the enzyme activity are selected to prepare immobilized glucose oxidase, and then the relative enzyme activity is measured, and the result shows that the optimal ratio of the carrier dosage to the enzyme activity is 1:500, so that 0.5 mL of enzyme solution immobilized by 1 g of resin is the optimal ratio of the carrier dosage to the enzyme activity (figure 5A).
Different glutaraldehyde concentrations from 0.5% to 5% are selected for immobilization of glucose oxidase, immobilized glucose oxidase is prepared, relative enzyme activity is measured, and the result shows that the optimal glutaraldehyde crosslinking concentration is 3% (fig. 5B).
The enzyme and resin combination needs a certain time, 5 different immobilization times from 5 h to 20 h are respectively selected, the immobilized glucose oxidase is prepared, the relative enzyme activity is measured, and the result shows that the optimal immobilization time is 10 h (figure 5C).
The temperature of the enzyme immobilization has an effect on the activity of the immobilized enzyme. The immobilization of glucose oxidase was performed at 5 different temperatures from 10 ℃ to 30 ℃ and then the relative enzyme activity was determined, showing that the effect was best at 25 ℃ (fig. 5D).
Example 4 Co-immobilization of glucose oxidase and Catalase
(1) Taking appropriate amount of amino resin, adding 100 mM K 2 HPO 4 -KH 2 PO 4 (pH value is 5.0) buffer solution, so that the resin is completely soaked in the buffer solution and activated for 5 hours at 25 ℃ and 120 rpm;
(2) placing the activated amino resin obtained in the step (1) into a buffer solution, adding a glutaraldehyde solution with the final concentration of 3%, crosslinking at 25 ℃ and 120 rpm for 5 h, and then using K 2 HPO 4 -KH 2 PO 4 Washing the glutaraldehyde-crosslinked resin with a buffer solution for 3 times to wash away residual glutaraldehyde solution to obtain modified resin;
(3) k for modifying the resin obtained in the step (2) 2 HPO 4 -KH 2 PO 4 Immersing buffer solution (pH value is 5.0), adding 500 units of glucose oxidase and catalase per gram of wet resin, and fixing at 120 rpm for 10 h at 25 ℃;
(4) filtering the immobilized enzyme obtained in the step (3) by a filter with the aperture of 100 mu m, removing the buffer solution containing the mixed enzyme solution, and then using the K at the temperature of 4 DEG C 2 HPO 4 -KH 2 PO 4 (pH 5.0) 3 times, filtering, and storing in a refrigerator at 4 deg.C.
Example 5 liquid enzymatic Co-production of fructose and gluconic acid
(1) Dissolving the crystallized D-glucose in purified water to prepare a 30% D-glucose solution; adjusting the pH value to 8.0 +/-0.5 by using a 5M NaOH solution;
(2) adding 10% of liquid glucose isomerase (equivalent to the mass of D-glucose) into the D-glucose solution obtained in the step (1), and carrying out catalytic reaction for 8 hours at 70 +/-0.5 ℃ and 100 rpm;
(3) heating the reaction liquid obtained in the step (2) to 100 ℃ and maintaining for 20min to inactivate enzyme; cooling to room temperature, centrifuging at 10000 rpm for 10 min, removing heat denaturation zymoprotein, and keeping supernatant;
(4) adjusting the pH of the centrifugal supernatant obtained in the step (3) to 4.5 by using 5M HCl, adding glucose oxidase and catalase with the use amounts of 10% and 6% (equivalent to the mass of D-glucose) respectively, and reacting for 8 hours at 55 ℃ and pH 4.5 at 100 rpm;
(5) heating the reaction liquid obtained in the step (4) to 100 ℃ and maintaining for 20min to inactivate enzyme; cooling to room temperature, centrifuging at 10000 rpm for 10 min, removing heat-denatured enzyme protein, and keeping supernatant;
(6) enabling the centrifugal supernatant obtained in the step (5) to flow through a resin column loaded with pretreated macroporous styrene series weak-base anion exchange resin D309, and adsorbing gluconic acid to obtain a D-fructose permeation solution with the content of 95%;
(7) desorbing the ion exchange resin column in the step (6) by using 0.5M dilute hydrochloric acid to obtain a gluconic acid solution.
Example 6 catalytic coproduction of fructose and calcium gluconate with immobilized enzyme
(1) Dissolving the crystallized D-glucose in purified water to prepare a 30% D-glucose solution; adjusting the pH value to 8.0 +/-0.5 by using a 5M NaOH solution;
(2) adding 5% immobilized glucose isomerase (equivalent to the mass of D-glucose) into the D-glucose solution obtained in the step (1), and carrying out catalytic reaction for 8 hours at 70 +/-0.5 ℃ and 100 rpm;
(3) centrifuging reaction liquid obtained in the step (2) at 8000 rpm for 5 min to remove the immobilized enzyme;
(4) adjusting the pH of the supernatant obtained in the step (3) to 4.5 by using 5M HCl, adding the immobilized glucose oxidase and catalase prepared in the example 2, wherein the using amount of the immobilized glucose oxidase and catalase is 20% (corresponding to the mass of the total sugar), and reacting for 8 hours at 55 ℃ and pH 4.5 at 100 rpm;
(5) centrifuging the reaction liquid obtained in the step (4) at 8000 rpm for 5 min to remove the immobilized enzyme;
(6) adding 0.5% powdered activated carbon into the centrifugal supernatant obtained in the step (5), adsorbing for 0.5 h at 50 ℃ and 100 rpm for decoloring, and filtering the activated carbon by using a 400-mesh filter;
(7) enabling the clear liquid obtained in the step (6) to flow through a resin column loaded with pretreated macroporous styrene series weak base anion exchange resin D309, and adsorbing gluconic acid to obtain a permeate containing D-fructose;
(8) desorbing the ion exchange resin column in the step (7) by using 0.5M dilute hydrochloric acid to obtain a gluconic acid solution;
(9) adding saturated Ca (OH) into the gluconic acid solution obtained in the step (8) 2 And (5) the pH value of the solution is neutral to obtain a calcium gluconate solution.
The above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions.

Claims (10)

1. A method for the enzymatic catalysis of glucose for the co-production of fructose and gluconic acid or gluconate, the method comprising the steps of:
(1) preparing a D-glucose solution by taking D-glucose as a raw material;
(2) adding the D-glucose solution obtained in the step (1) into glucose isomerase for catalytic reaction to obtain a reaction solution, centrifuging, and taking a supernatant;
(3) adding glucose oxidase and catalase into the supernatant obtained in the step (2), carrying out catalytic reaction to obtain a reaction solution, centrifuging, and taking the supernatant;
(4) enabling the supernatant obtained in the step (3) to flow through an adsorption resin column, and adsorbing gluconic acid to obtain a permeate containing D-fructose;
(5) desorbing the adsorption resin column in the step (4) by using an acid solution to obtain a gluconic acid solution;
(6) and (4) adding the gluconic acid solution obtained in the step (5) into an alkali solution containing metal ions until the pH is neutral to obtain a gluconic acid salt solution containing the metal ions.
2. The method for the enzyme-catalyzed glucose coproduction of fructose and gluconic acid or gluconate as claimed in claim 1, wherein the concentration of the D-glucose solution in the step (1) is 10% -80%, and the D-glucose solution is prepared by adding purified water to D-glucose crystals.
3. The method for the enzyme-catalyzed glucose coproduction of fructose and gluconic acid or gluconate as claimed in claim 1, wherein the glucose isomerase in step (2) is selected from a free or immobilized glucose isomerase in an amount of 1-30% by mass of the D-glucose solution.
4. The method for co-producing fructose and gluconic acid or gluconate by enzyme catalysis of glucose according to claim 1, wherein the conditions of the catalytic reaction in the step (2) are as follows: the catalysis pH is 7.0-9.0, the catalysis temperature is 65-75 ℃, the catalysis speed is 90-110 rpm, and the catalysis time is 4-10 h.
5. The method for co-producing fructose and gluconic acid or gluconate by enzyme catalysis of glucose according to claim 1, wherein in the step (3), the glucose oxidase is selected from free or immobilized glucose oxidase, and the dosage of the glucose oxidase is 1-30% of the mass of the D-glucose solution; the catalase is selected from free catalase or immobilized catalase, and the dosage of the catalase is 1-30% of the mass of the D-glucose solution.
6. The method for co-producing fructose and gluconic acid or gluconate by enzyme catalysis of glucose according to claim 1, wherein the conditions of the catalytic reaction in the step (3) are as follows: the catalysis pH is 4-5, the catalysis temperature is 50-60 ℃, the catalysis speed is 90-110 rpm, and the catalysis time is 3-8 h.
7. The method for co-producing fructose and gluconic acid or gluconate by using enzyme to catalyze glucose according to claim 1, wherein the adsorption resin column in the step (4) is an anion exchange resin column, specifically a macroporous styrene weak-base anion exchange resin column; the purity of the D-fructose in the permeate is not lower than 95%.
8. The method for co-producing fructose and gluconic acid or gluconate salt from glucose catalyzed by enzyme according to claim 1, wherein the acid solution in step (5) is diluted hydrochloric acid with a concentration of 0.5M.
9. The enzyme-catalyzed glucose co-production process of fructose and gluconic acid or gluconate as claimed in claim 1, wherein the metal ions in step (6) comprise potassium, sodium, calcium, iron, zinc, magnesium; the concentration of the alkali solution was 5M or a saturated solution at room temperature.
10. The method for co-producing fructose and gluconic acid or gluconate through enzyme catalysis by claim 5, wherein in the step (2) or (3), if immobilized glucose isomerase, glucose oxidase or catalase is selected, the reaction solution is not required to be heated for enzyme deactivation; if free glucose isomerase, glucose oxidase or catalase is selected, heating and enzyme deactivation treatment needs to be carried out on the reaction liquid, and the steps are as follows: heating the reaction solution to 100 ℃ and maintaining for 20min to inactivate the enzyme; cooling to room temperature, centrifuging at 8000-10000 rpm for 10-20 min, and collecting supernatant.
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