Preparation method of mannitol and method for regenerating immobilized enzyme carrier

Abstract

The invention provides a mannitol preparation method and an immobilized enzyme carrier regeneration method, and the method comprises the following steps: in the presence of divalent metal ions, taking fructose and a hydrogen source as reaction raw materials, and carrying out a reaction in the presence of sodium sulfide in a catalytic system composed of a coenzyme and an immobilized enzyme to obtain mannitol, wherein the immobilized enzyme is obtained by co-immobilizing mannitol dehydrogenase and formate dehydrogenase on a carrier.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to a method for preparing mannitol and a method for regenerating an immobilized enzyme carrier. Background Art

[0002] In recent years, the demand for D-mannitol as a functional sugar alcohol and low-calorie sweetener has continued to grow. It is an acyclic hexose alcohol, and D-sorbitol is its isomer, which is mostly in the form of white powder or needle-shaped solid. As a sugar substitute, the caloric value of D-mannitol is lower than that of most sugars, so D-mannitol can be widely used in food, chemical industry, clinical medicine and other fields. In the food field, mannitol can be used as a sweetener and anti-sticking agent for sugar-free products. In the medical field, mannitol is a commonly used drug that can prevent and treat acute renal failure and help to quickly clear toxins. At present, the world consumes more than 200,000 tons of D-mannitol each year, accounting for more than 11% of the total use of polyols (Hu Mengying, Zhang Tao, Research Progress in Microbial Fermentation Conversion of Mannitol, Food and Fermentation Industry, Vol. 46, No. 18, 2020). It is extremely important to find a method for preparing mannitol.

[0003] At present, the main method for producing D-mannitol in China is chemical synthesis, which requires high temperature and high pressure conditions. The use of Raney nickel catalytic hydrogenation can convert 50% (volume fraction) of fructose syrup into a mixture of about 30% mannitol and 70% sorbitol, and then obtain mannitol by low-temperature crystallization. The advantages of the chemical catalytic hydrogenation method are reliable process, high raw material utilization, and no geographical environment. The cost is low, but the disadvantage is that the high proportion of by-product sorbitol leads to a low mannitol yield. In addition, high temperature and high pressure reaction conditions are required, which will have certain safety issues. Due to the many defects of the chemical method, the production process of biological synthesis of D-mannitol is being continuously explored. The biological production process can not only promote the wider application of mannitol, but also has the potential to promote the development of the starch industry as a raw material (Wei, XL; Li, QZ; Hu, CC; You, C. An ATP-free in vitro synthetic enzymatic biosystem facilitating one-potstoichiometric conversion of starch to mannitol [J]. Applied Microbiology and Biotechnology, 2021, 105, 1913-1924.). There are many studies on the production of mannitol by microbial fermentation, and a large number of high-yield strains and corresponding fermentation processes have been developed. However, the production cost is high and the cycle is long.

[0004] The biocatalytic method uses fructose as a substrate. Under the catalysis of mannitol dehydrogenase (MDH), D-fructose is directly converted into D-mannitol. This process requires the participation of cofactors NADH or NADPH. The biocatalytic method also has certain limitations (Pan Shan, Research on the Efficient Conversion of Fructose to Prepare D-mannitol by Microbial Method. Jiangnan University, 2022, 000495): 1. The concentration of the catalytic substrate is limited, and the catalytic enzyme powder or enzyme solution can only be used once and cannot be reused; 2. The reported enzyme immobilization method has a low number of synthesized mannitol and has a low number of repetitions. The immobilized resin is relatively expensive. The simplified reaction formula of the biocatalytic method is as follows:

[0005]

[0006] In summary, it is very important to develop a method for preparing mannitol that has lower cost, is more environmentally friendly, has a high substrate concentration, allows for multiple reuses of the immobilized enzyme, and allows for repeated regeneration of the carrier. Summary of the invention

[0007] In view of the deficiencies in the prior art, the object of the present invention is to provide a method for preparing mannitol and a method for regenerating an immobilized enzyme carrier.

[0008] To achieve this object, the present invention adopts the following technical solutions:

[0009] In one aspect, the present invention provides a method for preparing mannitol, the method comprising the following steps:

[0010] In the presence of divalent metal ions, fructose and a hydrogen source are used as reaction raw materials, and a catalytic system consisting of a coenzyme and an immobilized enzyme is used to react in the presence of sodium sulfide to obtain mannitol; wherein the immobilized enzyme is obtained by co-immobilizing mannitol dehydrogenase and formate dehydrogenase on a carrier.

[0011] In the present invention, mannitol is prepared by using co-immobilized enzymes of mannitol dehydrogenase and formate dehydrogenase in the presence of sodium sulfide, with fructose and a hydrogen source as reaction raw materials. The co-immobilized enzymes used have high reuse rate, good stability, low cost, are more environmentally friendly, and have high substrate concentration, good catalytic effect, and high product yield.

[0012] Preferably, the divalent metal ion is Mg 2+ , Mn 2+ 、Zn 2+ or Ni 2+ wait.

[0013] Preferably, the concentration of the divalent metal ions in the reaction system is 1 mM to 10 mM, for example, 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM or 10 mM.

[0014] Preferably, the hydrogen source comprises sodium formate and / or formic acid.

[0015] Preferably, the amount of the hydrogen source is 1.0 to 1.5 molar equivalents of fructose, for example, 1.0 molar equivalent, 1.1 molar equivalent, 1.2 molar equivalent, 1.3 molar equivalent, 1.4 molar equivalent or 1.5 molar equivalent.

[0016] Preferably, the mass percentage concentration of fructose in the reaction system is 20% to 30%, for example, 20%, 23%, 25%, 28% or 30%.

[0017] Preferably, the coenzyme is coenzyme NAD + .

[0018] Preferably, the mass percentage concentration of the coenzyme in the system is 0.1% to 0.3%, for example, 0.1%, 0.15%, 0.18%, 0.2%, 0.23%, 0.25%, 0.28% or 0.3%.

[0019] Preferably, the mass ratio of mannitol dehydrogenase to formate dehydrogenase in the co-immobilized enzyme is (1-1.5):1, such as 1:1, 1.1:1, 1.2:1, 1.3:1, 1.4:1 or 1.5:1.

[0020] Preferably, the mass ratio of the total mass of the mannitol dehydrogenase and the formate dehydrogenase to the carrier is (2-4):1, for example, 2:1, 2.2:1, 2.4:1, 2.6:1, 2.8:1, 3:1, 3.3:1, 3.6:1, 3.8:1 or 4:1.

[0021] Preferably, the mass ratio of fructose to carrier is 1:(1-1.5), for example 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4 or 1:1.5.

[0022] Preferably, the carrier is an amino resin.

[0023] Preferably, the method for preparing the co-immobilized enzyme comprises the following steps:

[0024] The activated amino resin is added to a mixed enzyme solution of mannitol dehydrogenase and formate dehydrogenase, the pH of the system is adjusted to 7.0-8.5 (e.g., 7.0, 7.4, 7.8, 8.0, 8.2 or 8.5), and then immobilized at 20°C-30°C (e.g., 20°C, 23°C, 25°C, 28°C or 30°C) and 150-250rpm (e.g., 150rpm, 160rpm, 180rpm, 200rpm, 230rpm or 250rpm).

[0025] Preferably, the pH of the system is adjusted using a Tris·HCl buffer solution with a pH of 8 and a concentration of 1 M.

[0026] Preferably, the immobilization time is 15 to 20 h (eg, 15 h, 16 h, 17 h, 18 h, 19 h or 20 h).

[0027] In the present invention, the amino resin is activated by using a homobifunctional reagent to activate the amino resin.

[0028] Preferably, the homobifunctional reagent comprises any one of glutaraldehyde, dextran or dialdehyde cellulose, more preferably glutaraldehyde.

[0029] In the present invention, 4 mL of pH 8.550 mM sodium phosphate buffer containing 2% of the same bifunctional reagent is added per gram of amino resin, and the resin is activated by shaking in a water bath at 25° C. for 1.5 hours. After activation, the resin changes from white to light orange, and the activated resin is washed with pure water for more than six times.

[0030] In the present invention, after the immobilization, the immobilized product can be washed with pure water for more than 4 times, and the supernatant enzyme solution after the immobilization can be taken and the protein content thereof can be determined by the Coomassie Brilliant Blue method to calculate the protein activity of the immobilized enzyme.

[0031] Preferably, the amount of sodium sulfide is 0.02% to 0.05% of the substrate mass, such as 0.02%, 0.03%, 0.04% or 0.05%. In the present invention, sodium sulfide can improve the stability of the immobilized enzyme, so that the number of applications of the immobilized enzyme can be significantly increased.

[0032] Preferably, the solvent of the reaction is Tris·HCl buffer solution, PBS buffer solution or purified water.

[0033] Preferably, the reaction is carried out at a pH of 6.5 to 7.5, for example a pH of 6.5, 6.7, 6.8, 7.0, 7.2, 7.4 or 7.5.

[0034] Preferably, the pH of the reaction system is regulated by formic acid.

[0035] Preferably, the reaction temperature is 30-40°C, for example 30°C, 33°C, 35°C, 38°C or 40°C, and the reaction time is 12-22h, for example 12h, 14h, 16h, 18h, 20h or 22h.

[0036] In the present invention, the immobilized enzyme can be recycled, and during the recycling reaction, the concentration of the substrate fructose can be 30%.

[0037] On the other hand, the present invention provides a method for regenerating an immobilized enzyme carrier, which comprises: treating the immobilized enzyme resin carrier used in the preparation method of mannitol as described above with an alkali solution for a first time and ultrasonicating, washing, then treating with an acid, washing, and then treating with an alkali solution for a second time, washing, to complete the regeneration of the immobilized enzyme carrier.

[0038] Preferably, the alkali solution for the first treatment and the second treatment is independently aqueous ammonia or sodium hydroxide solution, preferably sodium hydroxide solution.

[0039] Preferably, the concentration of the alkali solution is 2% to 5%, for example, 2%, 2.5%, 3%, 3.5%, 4%, 4.5% or 5%.

[0040] Preferably, the first treatment with alkaline solution is carried out at room temperature, and the treatment time is 2 h to 4 h, for example, 2 h, 2.5 h, 3 h, 3.5 h or 4 h.

[0041] Preferably, the second treatment with alkaline solution is carried out at room temperature, and the treatment time is 2 h to 4 h, for example, 2 h, 2.5 h, 3 h, 3.5 h or 4 h.

[0042] Preferably, the acid used in the acid treatment is hydrochloric acid or sulfuric acid.

[0043] Preferably, the mass percentage concentration of the acid is 10% to 20%, for example, 10%, 12%, 14%, 15%, 17%, 18% or 20%.

[0044] Preferably, the acid treatment is carried out at a temperature of 80°C to 100°C (e.g., 80°C, 85°C, 88°C, 90°C, 93°C, 95°C, 98°C or 100°C), and the treatment time is 12h to 24h, for example, 12h, 14h, 16h, 18h, 20h, 22h or 24h.

[0045] Preferably, the washing is carried out using water.

[0046] Compared with the prior art, the present invention has the following beneficial effects:

[0047] The invention uses co-immobilized enzymes of mannitol dehydrogenase and formate dehydrogenase in the presence of sodium sulfide, with fructose and a hydrogen source as reaction raw materials, to prepare mannitol. The co-immobilized enzymes used have high reuse rate, good stability, low cost, are more environmentally friendly, have high substrate concentration, good catalytic effect, and high product yield.

[0048] The co-immobilized enzyme resin carrier used multiple times in the present invention can be regenerated for repeated use, and the immobilized enzyme formed after the regeneration still has a high catalytic efficiency and can be repeatedly used, with good stability and high catalytic efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 This is the HPLC spectrum of standard mannitol.

[0050] Figure 2 This is the HPLC spectrum of the product after 12h reaction in Example 1.

[0051] Figure 3 The HPLC spectrum of sorbitol, a by-product produced in Example 1. DETAILED DESCRIPTION

[0052] The technical solution of the present invention is further described below by specific implementation methods. It should be understood by those skilled in the art that the embodiments are only used to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0053] Preparation Example 1

[0054] Preparation of immobilized enzyme:

[0055] (1) Activation of amino resin:

[0056] 4 ml of pH 8.5 50 mM sodium phosphate buffer containing 2% homobifunctional reagent glutaraldehyde was added per gram of amino resin, and activated by shaking in a water bath at 25°C for 1.5 h. After activation, the resin changed from white to light orange, and the activated resin was washed with pure water six times.

[0057] (2) Immobilization process:

[0058] The activated amino resin (model: Lanxiao LX-701) was added to the mixed enzyme solution of mannitol dehydrogenase (MDH, purchased from Merck reagent, item number F886125, enzyme activity ≥ 60u / g) and formate dehydrogenase (FDH, purchased from Merck reagent, item number M920972-5U, enzyme activity ≥ 60u / g), the mass ratio of the two enzymes was 1:1, and the mass ratio of the total mass of the mannitol dehydrogenase and formate dehydrogenase to the carrier was 3:1. The immobilization system pH was adjusted to 7.2 using Tris·HCl buffer solution (pH = 8, 1M), and the immobilization reaction was started at 200pm, 25°C, and the immobilization was carried out for 20h.

[0059] (3) Post-immobilization treatment:

[0060] The immobilized carrier was filtered out and washed with pure water for 4 times. The supernatant enzyme solution after immobilization was taken and the protein content was determined by Coomassie Brilliant Blue method. The enzyme protein immobilization efficiency was calculated to be 93%.

[0061] Preparation Example 2

[0062] Preparation of immobilized enzyme:

[0063] The amino resin activation was the same as in Preparation Example 1.

[0064] Immobilization process: (1) Add the activated amino resin (model: Lanxiao LX-701) to the mannitol dehydrogenase (MDH purchased from Merck Reagent, product number F886125, enzyme activity ≥ 60u / g) enzyme solution, the mass ratio of the mannitol dehydrogenase mass to the carrier is 1.5:1. Use Tris·HCl buffer solution (pH=8, 1M) to adjust the pH of the immobilization system to 7.2, start the immobilization reaction at 200pm, 25°C, and immobilize for 20h; filter out the immobilized carrier and wash it with pure water 4 times, and take the supernatant enzyme solution after immobilization, and determine the protein content therein by the Coomassie Brilliant Blue method. The enzyme protein immobilization efficiency is calculated to be 93%, and the obtained immobilized enzyme is recorded as immobilized enzyme 1;

[0065] (2) The activated amino resin (model: Lanxiao LX-701) was added to the formate dehydrogenase (FDH, purchased from Merck Reagent, catalog number M920972-5U, enzyme activity ≥ 60u / g) enzyme solution, the mass ratio of the formate dehydrogenase to the carrier was 1.5:1, and the mass ratio of the fructose to the carrier was 1:1.5. The immobilization system pH was adjusted to 7.2 using Tris·HCl buffer solution (pH=8, 1M), and the immobilization reaction was started at 200pm and 25°C for 20 hours; the immobilized carrier was filtered out and washed with pure water 4 times, and the supernatant enzyme solution after immobilization was taken, and the protein content thereof was determined by the Coomassie Brilliant Blue method. The enzyme protein immobilization efficiency was calculated to be 94%, and the obtained immobilized enzyme was recorded as immobilized enzyme 2.

[0066] Example 1

[0067] This example investigates the effect of reaction temperature on the catalytic synthesis of mannitol.

[0068] Substrate fructose 30g, sodium formate as substrate 0.3 equivalent, 0.015g sodium sulfide, manganese chloride concentration of 5mM, coenzyme NAD + 0.03g, added to 80mL Tris·HCl buffer solution, stirred evenly, controlled pH to 6.5, prepared in Example 1, the co-immobilized enzyme (wherein the carrier mass is 40g) was loaded into a chromatography column (manufacturer: Tianyan Biology, EZ type: 55ml protein purification chromatography empty column, the same below) and connected to the reaction system with a hose, turned on the peristaltic pump (pump speed 15rpm) to make the system circulate for reaction, pH was added dropwise by an automatic titrator (15% formic acid regulation), and the amount of formic acid added was 0.8 equivalents of the substrate. The reaction system temperature was set to 25°C, 30°C, 35°C, 40°C, 45°C, and the conversion rates corresponding to the reaction for 12h were 79.12%, 96.28%, 99.82%, 97.46%, and 85.26%, respectively.

[0069] The product was determined by HPLC (Agilent 1260II with differential detector; method: mobile phase: water, flow rate 1 ml / min, isocratic, running time 30 min, injection volume 10 μL) and compared with mannitol standard. Figure 1 is the HPLC spectrum of standard mannitol, Figure 2 The HPLC spectrum of the product of the present embodiment after 12h reaction (the product spectra produced at different temperatures are consistent), it can be seen that the product produced by the present invention is mannitol. And the byproduct sorbitol produced in this embodiment has an HPLC spectrum as shown in FIG. Figure 3 shown.

[0070] Example 2

[0071] This example investigates the effect of reaction pH on the catalytic synthesis of mannitol.

[0072] Substrate fructose 30g, sodium formate as substrate 0.3 equivalent, 0.015g sodium sulfide, manganese chloride concentration of 5mM, coenzyme NAD + 0.03g was added to 80mL Tris·HCl buffer solution, stirred evenly, the pH was controlled to the corresponding value, and the temperature of the reaction system was set to 35°C. The co-immobilized enzyme prepared in Preparation Example 1 (wherein the carrier mass was 40g) was loaded into the chromatography column and connected to the reaction system, and the peristaltic pump (pump speed 15rpm) was turned on to circulate the system for reaction, and the pH was added dropwise by an automatic titrator (15% formic acid regulation), and the amount of formic acid added was 0.8 equivalents of the substrate. The pH of the reaction system was set to 6.0, 6.5, 7.0, and 7.5, respectively. The conversion rates corresponding to the reaction for 12h were 50.12%, 99.82%, 97.34%, and 82.62%, respectively.

[0073] Example 3

[0074] This example investigates the effect of the type of enzyme activator on the catalytic synthesis of mannitol.

[0075] Substrate fructose 30g, sodium formate as substrate 0.3 equivalent, 0.015g sodium sulfide, add 5mM metal ion inorganic salt to the system, coenzyme NAD +0.03g, added to 80mL Tris·HCl buffer solution, stirred evenly, controlled pH to 6.5, the temperature of the reaction system was set to 35°C. The co-immobilized enzyme prepared in Preparation Example 1 (wherein the carrier mass is 40g) was loaded into a chromatographic column and connected to the reaction system, the peristaltic pump was turned on (pump speed 15rpm) to circulate the system for reaction, and the pH was added dropwise by an automatic titrator (15% formic acid regulation), and the amount of formic acid added dropwise was 0.8 equivalents of the substrate. The added metal ion inorganic salts were magnesium chloride, manganese chloride, zinc chloride and nickel chloride, respectively. The conversion rates corresponding to the reaction for 12h were 96.62%, 99.82%, 97.24% and 95.43%, respectively.

[0076] Example 4

[0077] This example investigates the effect of a homobifunctional reagent on the catalytic synthesis of mannitol.

[0078] Substrate fructose 30g, sodium formate as substrate 0.3 equivalent, 0.015g sodium sulfide, manganese chloride concentration of 5mM, 0.1% coenzyme NAD + 0.03g, add 80mL Tris·HCl buffer solution, stir evenly, control pH to 6.5, and set the temperature of the reaction system to 35°C. The co-immobilized enzyme (of which the carrier mass is 40g) is loaded into the chromatography column and connected to the reaction system, and the pH is added dropwise by an automatic titrator (15% formic acid adjustment), and the amount of formic acid added is 0.8 equivalents of the substrate.

[0079] The co-immobilized enzyme was prepared by the method in Preparation Example 1, and in the preparation process, glutaraldehyde, dextran and dialdehyde cellulose were used as homobifunctional reagents (2% of the mass of the enzyme solution).

[0080] The conversion rates of preparing mannitol after 12 h of reaction were 99.82%, 96.42% and 97.38% respectively.

[0081] Comparative Example 1

[0082] This comparative example illustrates the effect of using hydrochloric acid to regulate pH on the enzyme-catalyzed synthesis of mannitol.

[0083] Substrate fructose 30g, sodium formate as substrate 1.1 equivalents, 0.015g sodium sulfide, manganese chloride concentration of 5mM, 0.1% coenzyme NAD + 0.03g, add 80mL Tris·HCl buffer solution, stir evenly, control the pH to 6.5, and set the temperature of the reaction system to 35°C. The co-immobilized enzyme prepared in Preparation Example 1 (wherein the carrier mass is 40g) is loaded into the chromatography column and connected to the reaction system, and the peristaltic pump (pump speed 15rpm) is turned on to circulate the system for reaction, and the pH is added dropwise by an automatic titrator (15% hydrochloric acid is adjusted), and the conversion rate corresponding to the reaction for 12h is 85.24%.

[0084] Comparative Example 2

[0085] The immobilized enzyme 1 prepared in Preparation Example 2 was mixed with the immobilized enzyme 2 to prepare the immobilized enzyme.

[0086] Substrate fructose 30g, sodium formate as substrate 0.3 equivalent, 0.015g sodium sulfide, manganese chloride concentration of 5mM, 0.1% coenzyme NAD + 0.03g, add 80mL Tris·HCl buffer solution, stir evenly, control pH to 6.5, and set the temperature of the reaction system to 35°C. Preparation Example 2 The immobilized enzyme 1 (wherein the carrier mass is 20g) prepared was mixed with the immobilized enzyme 2 (wherein the carrier mass is 20g) as the immobilized enzyme and loaded into the chromatography column and connected to the reaction system, and the peristaltic pump (pump speed 15rpm) was turned on to circulate the system for reaction, and the pH was added dropwise by an automatic titrator (15% formic acid adjustment), and the amount of formic acid added was 0.8 equivalents of the substrate, and the conversion rate corresponding to the reaction for 12h was 88.6%.

[0087] Example 5

[0088] This example examines the preparation of mannitol using a 30% substrate concentration.

[0089] Substrate fructose 270g, sodium formate as substrate 0.3 equivalent, 0.135g sodium sulfide, manganese chloride concentration of 5mM, coenzyme NAD + 0.27g, add 720mL Tris·HCl buffer solution, stir evenly, control pH to 6.5, and set the temperature of the reaction system to 35°C. The co-immobilized enzyme prepared in Preparation Example 1 (wherein the carrier mass is 360g) is loaded into a chromatography column and connected to a reaction system, and the pH is added dropwise by an automatic titrator (15% formic acid adjustment), and the amount of formic acid added is 0.8 equivalents of the substrate. The conversion rate corresponding to 12h of reaction is 99.24%, indicating that the enzyme-catalyzed reaction system of the present invention has good applicability to high-concentration substrates.

[0090] Example 6

[0091] This example investigates the stability of the immobilized enzyme.

[0092] Substrate fructose 30g, sodium formate as substrate 0.3 equivalent, 0.015g sodium sulfide, manganese chloride concentration of 5mM, 0.1% coenzyme NAD +0.03g, added to 80mL Tris·HCl buffer solution, stirred evenly, controlled pH to 6.5, and the temperature of the reaction system was set to 35°C. The co-immobilized enzyme (40g) prepared in Preparation Example 1 was loaded into a chromatography column and connected to a reaction system, and the peristaltic pump (pump speed 15rpm) was turned on to allow the system to circulate for reaction, and the pH was added dropwise by an automatic titrator (15% formic acid adjustment), and the amount of formic acid added was 0.8 equivalents of the substrate. The reaction was cyclically reacted 110 times, and the conversion rate is shown in Table 1 below.

[0093] Table 1

[0094]

[0095] Comparative Example 3

[0096] This comparative example illustrates the effect of not adding sodium sulfide on the stability of the catalytic synthesis of mannitol.

[0097] Substrate fructose 30g, sodium formate as substrate 0.3 equivalent, manganese chloride concentration 5mM, 0.1% coenzyme NAD + 0.03g, added to 80mL Tris·HCl buffer solution, stirred evenly, controlled pH to 6.5, the reaction system temperature was set to 35°C. Preparation Example 1 The co-immobilized enzyme (40g) prepared was immobilized and loaded into a chromatography column and connected to the reaction system, the peristaltic pump (pump speed 15rpm) was turned on to make the system circulate for reaction, the pH was added dropwise by an automatic titrator (15% formic acid adjustment), and the amount of formic acid added was 0.8 equivalents of the substrate. The cycle reaction was repeated 80 times, and the conversion rate is shown in Table 2.

[0098] Table 2

[0099]

[0100] From the results, it can be seen that when sodium sulfide is not added to the reaction system, the stability of the immobilized enzyme decreases. After being applied for more than 80 times, the conversion rates from the 81st to the 90th times are 89.28% to 81.62%, showing a clear downward trend.

[0101] Example 7

[0102] This example investigates the effect of sulfuric acid treatment at different temperatures on the number of regeneration resin cycle reactions.

[0103] 50 g of the resin carrier of Example 6 which has been recycled 110 times was added with 100 g of 5% sodium hydroxide solution at room temperature, ultrasonicated for 1 hour, stirred at room temperature for 20 hours, and washed with water for 5 times; 100 g of 20% sulfuric acid solution was added, stirred at 30°C, 50°C, 80°C, and 100°C for 24 hours, and washed with water for 5 times; finally, 100 g of 5% sodium hydroxide solution was added at room temperature, stirred for 4 hours, and washed with water until it became weakly alkaline to obtain a regenerated resin carrier.

[0104] The enzyme was immobilized on the regenerated resin support according to Preparation Example 1, and the stability of the enzyme immobilized on the regenerated resin support was further investigated according to the method of Example 6. When the reaction conversion rate of the cyclic reaction was lower than 90%, the cyclic reaction was stopped. The results showed that the regenerated resin was used 6 times, 15 times, 28 times, and 24 times, respectively.

[0105] Example 8

[0106] This example investigates the effect of different acid concentrations on the number of regeneration resin cycle reactions.

[0107] 50 g of the resin carrier of Example 6 which has been recycled 110 times was added with 100 g of 5% sodium hydroxide solution at room temperature, ultrasonicated for 1 hour, stirred at room temperature for 20 hours, and washed with clean water 5 times; 100 g of 5%, 10%, 15%, and 20% sulfuric acid solutions were added respectively, stirred at 80°C for 24 hours, and washed with clean water 5 times; finally, 100 g of 5% sodium hydroxide solution was added and stirred for 4 hours, and washed with clean water until it became weakly alkaline to obtain a regenerated resin carrier.

[0108] The enzyme was immobilized on the regenerated resin support according to Preparation Example 1, and the stability of the enzyme immobilized on the regenerated resin support was further investigated according to the method of Example 6. When the reaction conversion rate of the cyclic reaction was lower than 90%, the cyclic reaction was stopped. The results showed that the regenerated resin was used 12 times, 21 times, 26 times, and 28 times, respectively.

[0109] Example 9

[0110] This example investigates the effects of different types of alkali treatment on the number of resin regeneration cycle reactions.

[0111] 50 g of the resin carrier of Example 6 which has been recycled 110 times was respectively added with 100 g of 5% ammonia solution and 100 g of 5% sodium ethoxide solution at room temperature, ultrasonicated for 1 h, stirred at room temperature for 20 h, and washed with water 5 times; 100 g of 20% sulfuric acid solution was added, stirred at 80°C for 24 h, and washed with water 5 times; finally, 100 g of 5% ammonia solution and 100 g of 5% sodium ethoxide solution were respectively added, stirred for 4 h, and washed with water until it became weakly alkaline to obtain a regenerated resin carrier.

[0112] The enzyme was immobilized on the regenerated resin support according to Preparation Example 1, and the stability of the enzyme immobilized on the regenerated resin support was further investigated according to the method of Example 6. When the reaction conversion rate of the cyclic reaction was lower than 90%, the cyclic reaction was stopped. The results showed that the regenerated resin was used 18 times and 10 times, respectively.

[0113] Example 10

[0114] This example investigates the effect of regenerating a used resin on the number of cyclic reactions of the immobilized enzyme.

[0115] To regenerate 50 g of the resin carrier of Example 8 that has been used 28 times, add 100 g of 5% sodium hydroxide solution at room temperature, ultrasonicate for 1 hour, continue stirring at room temperature for 20 hours, and wash with clean water 5 times; add 100 g of 20% sulfuric acid solution, stir at 80°C for 24 hours, and wash with clean water 5 times; finally, add 100 g of 5% sodium hydroxide solution at room temperature, stir for 4 hours, and wash with clean water until it becomes weakly alkaline to obtain a regenerated resin carrier.

[0116] The enzyme was immobilized on the regenerated resin support according to Preparation Example 1, and the stability of the enzyme immobilized on the regenerated resin support was further investigated according to the method of Example 6. When the reaction conversion rate of the cyclic reaction was lower than 90%, the cyclic reaction was stopped. The results showed that the regenerated resin was used for 19 times.

[0117] The applicant declares that the present invention illustrates the preparation method of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned embodiments, that is, it does not mean that the present invention must rely on the above-mentioned embodiments to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of various raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. A method for preparing mannitol, characterized in that: The method comprises the following steps: In the presence of divalent metal ions, fructose and a hydrogen source are used as reaction raw materials, and a catalytic system consisting of a coenzyme and an immobilized enzyme is used to react in the presence of sodium sulfide to obtain mannitol; wherein the immobilized enzyme is obtained by co-immobilizing mannitol dehydrogenase and formate dehydrogenase on a carrier.

2. The method according to claim 1, characterized in that The divalent metal ion is Mg 2+ , Mn 2+ 、Zn 2+ or Ni 2+ ; Preferably, the concentration of the divalent metal ions in the reaction system is 1 mM to 10 mM.

3. The method according to claim 1 or 2, characterized in that: The hydrogen source includes sodium formate and / or formic acid; Preferably, the amount of the hydrogen source is 1.0 to 1.5 molar equivalents of fructose; Preferably, the mass percentage concentration of fructose in the reaction system is 20% to 30%; Preferably, the coenzyme is coenzyme NAD + ; Preferably, the mass percentage concentration of the coenzyme in the system is 0.1% to 0.3%.

4. The method according to any one of claims 1 to 3, characterized in that The mass ratio of mannitol dehydrogenase to formate dehydrogenase in the co-immobilized enzyme is (1-1.5):1; Preferably, the mass ratio of the total mass of the mannitol dehydrogenase and the formate dehydrogenase to the carrier is (2-4):1; Preferably, the mass ratio of fructose to carrier is 1:(1-1.5).

5. The method according to any one of claims 1 to 4, characterized in that The carrier is an amino resin; Preferably, the method for preparing the co-immobilized enzyme comprises the following steps: The activated amino resin is added to the mixed enzyme solution of mannitol dehydrogenase and formate dehydrogenase, the pH of the system is adjusted to 7.0-8.5, and then immobilized at 20-30°C and 150-250rpm; Preferably, the immobilization time is 15 h to 20 h.

6. The method according to claim 5, characterized in that The activation of the amino resin is to activate the amino resin using a homobifunctional reagent; Preferably, the homobifunctional reagent comprises any one of glutaraldehyde, dextran or dialdehyde cellulose, more preferably glutaraldehyde.

7. The method according to any one of claims 1 to 6, characterized in that The amount of sodium sulfide used is 0.02% to 0.05% of the substrate mass; Preferably, the solvent of the reaction is Tris·HCl buffer solution, PBS buffer solution or purified water; Preferably, the reaction is carried out at pH 6.5 to 7.5; Preferably, the pH of the reaction system is regulated by formic acid; Preferably, the reaction temperature is 30-40° C., and the reaction time is 12-22 hours.

8. A method for regenerating an immobilized enzyme carrier, characterized in that: The method comprises: The immobilized enzyme resin carrier used in the method according to any one of claims 1 to 7 is treated with alkali solution for the first time and ultrasonically washed, then treated with acid and washed, and then treated with alkali solution for the second time and washed to complete the regeneration of the immobilized enzyme carrier.

9. The method according to claim 8, characterized in that The alkali solution for the first treatment and the second treatment is independently ammonia water or sodium hydroxide solution, preferably sodium hydroxide solution; Preferably, the concentration of the alkali solution is 2% to 5%.

10. The method according to claim 8, characterized in that The first treatment with alkali solution is carried out at room temperature for 2 to 4 hours; Preferably, the second treatment with alkaline solution is carried out at room temperature for 2 to 4 hours; Preferably, the acid used in the acid treatment is hydrochloric acid or sulfuric acid; Preferably, the mass percentage concentration of the acid is 10% to 20%; Preferably, the acid treatment is carried out at a temperature of 80°C to 100°C and the treatment time is 12h to 24h; Preferably, the washing is carried out using water.

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