Immobilized enzyme, preparation method and application thereof, and preparation method of inositol

Abstract

The invention discloses an immobilized enzyme, a preparation method and application thereof and a preparation method of inositol, and belongs to the technical field of enzyme catalysis. The immobilized enzyme takes amino resin as a carrier, the carrier is immobilized with enzymes, the enzymes comprise alpha-glucan phosphorylase, phosphoglucomutase, inositol-3-phosphate synthase and inositol monophosphate, the immobilized enzyme is used for preparing the inositol, and the method has the advantages that the time for preparing the inositol is short, the concentration of the generated inositol is high, the conversion rate is high, and the like. In addition, the immobilized enzyme can be repeatedly used, so that the method has the advantage of remarkably reducing the production cost of the inositol.

Description

Technical Field

[0001] The invention relates to the technical field of enzyme catalysis, and in particular to an immobilized enzyme and a preparation method and application thereof, and a method for preparing inositol. Background Art

[0002] Inositol is a six-carbon cyclic alcohol and a member of the water-soluble vitamin B family. It is a precursor of many intracellular components, such as phosphoinositides, phytic acid, phosphatidylinositol, phosphatidylinositol phosphate, inositol-containing sphingolipids and glycosylphosphatidylinositol membrane proteins. Due to its multiple biological functions, inositol is widely used in food, feed, pharmaceutical and cosmetic industries. Inositol plays a very important role in the regulation of sugar metabolism, chromatin and cytoskeleton remodeling, gene transcription, proliferation and apoptosis. Inositol is traditionally prepared by acid hydrolysis of phytic acid isolated from corn steep liquor and rice bran. However, this traditional process has the disadvantages of serious phosphorus pollution, harmful gas emissions and high energy consumption.

[0003] The construction of an in vitro enzyme synthesis pathway for inositol is the latest research and development hotspot. Using starch or maltodextrin as raw materials to synthesize inositol is an effective pathway that contains four heat-resistant enzymes: α-glucan phosphorylase (α-GP), phosphoglucomutase (PGM), inositol-3-phosphate synthase (IPS) and inositol monophosphatase (IMP). The synthesis process of inositol includes four stages. First, glucan phosphorylase converts a glucose unit in starch or starch derivatives into glucose-1-phosphate; second, glucose-1-phosphate is converted into glucose-6-phosphate by phosphoglucomutase; then, glucose-6-phosphate is converted into inositol-3-phosphate by inositol-3-phosphate synthase; and finally, inositol-3-phosphate is converted into inositol by inositol monophosphatase. Since the two enzyme-catalyzed reactions of inositol-3-phosphate synthase converting glucose-6-phosphate into inositol-3-phosphate and inositol-3-phosphate into inositol by inositol monophosphatase are irreversible reactions, the enzyme-catalyzed system can achieve a very high conversion rate. In addition, since starch is a mixture of straight-chain starch (20% to 30%) and amylopectin (70% to 80%), and the branches in amylopectin are connected to the main chain by α-1,6 glycosidic bonds, but glucan phosphorylase cannot decompose α-1,6 glycosidic bonds, in order to improve the conversion rate of inositol, a debranching enzyme, isoamylase, which can decompose α-1,6 glycosidic bonds in starch, is added to the multi-enzyme reaction system. The biosynthesis method described above is a green method for producing inositol, which is characterized by the high selectivity of multiple enzymes to bring high yields of inositol and easy product separation.

[0004] However, the enzymatic method for preparing inositol also has the disadvantages of high production cost of enzyme and unstable properties. In order to overcome the shortcomings of this type of enzyme in the application process, the immobilization technology of enzyme arises at the historic moment. This technology is a technology that utilizes a carrier to constrain the enzyme in a fixed area to carry out catalytic reaction and can be reused. It can not only improve the stability of the enzyme, but also promote the separation of the enzyme from the product and the reuse of the enzyme, thereby reducing the use cost of the enzyme. However, the existing reported immobilization method has the disadvantages of low generation concentration, high cost and long reaction time for generating inositol, which is not suitable for industrial application. Summary of the invention

[0005] In view of the technical problems of low inositol production concentration, high cost and long reaction time in the existing inositol preparation technology, the present application proposes an immobilized enzyme and its preparation method, application and inositol preparation method.

[0006] According to one aspect of the present invention, an immobilized enzyme is provided, using amino resin as a carrier, wherein the carrier is immobilized with enzymes, including α-glucan phosphorylase, phosphoglucomutase, inositol-3-phosphate synthase and inositol monophosphatase.

[0007] Furthermore, the amino resin is LX-1000HA resin.

[0008] Furthermore, the mass ratio of the α-glucan phosphorylase, phosphoglucomutase, inositol-3-phosphate synthase and inositol monophosphatase to the carrier is (1-1.5): (1-1.5): (3-5): (1-1.5): 1, preferably (1-1.5): (1-1.5): (4-5): (1-1.5): 1.

[0009] According to a second aspect of the present invention, a method for preparing an immobilized enzyme is provided, the method comprising:

[0010] S1. Activation of the carrier: adding 1,6-hexanediol diglycidyl ether and glutaraldehyde to the amino resin in sequence to react to obtain an activated carrier;

[0011] S2. Enzyme immobilization: reacting the enzyme solution composition with the activated carrier obtained in step S1 to obtain the immobilized enzyme, wherein the enzyme solution composition contains α-glucan phosphorylase, glucose phosphomutase, inositol-3-phosphate synthase and inositol monophosphatase.

[0012] Furthermore, the reaction time of adding 1,6-hexanediol diglycidyl ether for reaction in step S1 is 12 to 48 hours, preferably 24 hours; the reaction time of adding glutaraldehyde for reaction is 5 to 20 hours, preferably 10 hours.

[0013] Furthermore, the mass ratio of the 1,6-hexanediol diglycidyl ether to the carrier is 1:1 to 1:2; the mass ratio of the glutaraldehyde to the carrier is 1:8 to 1:12.

[0014] Furthermore, the method further comprises placing the immobilized enzyme obtained in step S2 in a glycine solution for reaction.

[0015] Furthermore, the pH of the glycine solution is 7 to 9, preferably 8.5; the reaction time is 18 to 30 hours, and the reaction temperature is 20 to 28° C.; preferably, the mass ratio of the glycine to the carrier is 1:1 to 1.5:1.

[0016] 1,6-hexanediol diglycidyl ether and glutaraldehyde are sequentially added to the carrier, so that the carrier has arms with epoxy groups and aldehyde groups, thereby enhancing the catalytic activity and stability of the immobilized enzyme.

[0017] The immobilized enzyme is post-treated with glycine so that the excess aldehyde and epoxy groups in the carrier react with glycine to avoid reacting with the active site of the enzyme and reducing the enzyme activity. At the same time, after the reaction of glycine with the excess aldehyde and epoxy groups, a hydrophilic microenvironment is formed around the enzyme, which is beneficial to maintaining the catalytic activity of the immobilized enzyme.

[0018] According to the third aspect of the present invention, a use of the immobilized enzyme according to the first aspect of the present invention or the immobilized enzyme obtained by the preparation method according to the second aspect of the present invention in catalyzing the preparation of inositol is provided.

[0019] According to a fourth aspect of the present invention, a method for preparing inositol is proposed, comprising the following steps: subjecting maltodextrin to an enzymatic reaction in the presence of the immobilized enzyme to obtain inositol; preferably, the mass ratio of the maltodextrin to the immobilized enzyme is 1:1 to 2:1, the reaction temperature of the enzymatic reaction is 65 to 75°C, and the reaction time is 60 to 80 hours.

[0020] Beneficial effects of the present invention:

[0021] Compared with the prior art, the present invention provides an immobilized enzyme and a preparation method and application thereof, which have good catalytic activity and stability. The present invention provides a method for preparing inositol using the immobilized enzyme, which can effectively shorten the time for preparing inositol and improve the inositol concentration and conversion rate. At the same time, the immobilized enzyme can be reused in the preparation of inositol, thereby having the advantage of reducing the production cost of inositol. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The accompanying drawings are included to provide a further understanding of the embodiments and are incorporated into and constitute a part of this specification. The accompanying drawings illustrate the embodiments and are used together with the description to explain the principles of the present invention. It will be easy to recognize other embodiments and many expected advantages of the embodiments because they become better understood by reference to the following detailed description. The elements of the drawings are not necessarily to scale with each other. The same reference numerals refer to corresponding similar parts.

[0023] Figure 1 The schematic diagram of the process for preparing the immobilized enzyme of the present invention is shown. DETAILED DESCRIPTION

[0024] The present application will be further described in detail below in conjunction with the accompanying drawings and embodiments. It is to be understood that the specific embodiments described herein are only used to explain the relevant invention, rather than to limit the invention. It should also be noted that, for ease of description, only the parts related to the relevant invention are shown in the accompanying drawings.

[0025] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present application can be combined with each other. The present application will be described in detail below with reference to the accompanying drawings and in combination with the embodiments.

[0026] The first object of the present invention is to provide an immobilized enzyme and a preparation method thereof, wherein amino resin is selected as a carrier, and the immobilized enzyme prepared has high enzyme activity and can still achieve a high inositol conversion rate when reused 6 times, with a conversion rate greater than 70%.

[0027] In view of the technical problems of low inositol production concentration and long reaction time in the existing inositol preparation technology, the second purpose of the present invention is to propose an application of the immobilized enzyme or the immobilized enzyme obtained according to the preparation method in catalyzing the preparation of inositol.

[0028] The third object of the present invention is to provide a method for preparing inositol, which can effectively shorten the time for preparing inositol and increase the concentration of the generated inositol.

[0029] The immobilized enzyme provided by the present invention includes an amino resin as a carrier, and the amino resin includes but is not limited to LX-1000HA resin (manufacturer: Xi'an Lanxiao Technology New Materials Co., Ltd.). The schematic flow diagram of the method for preparing the immobilized enzyme in the present invention is as follows Figure 1 As shown, it is prepared according to the following method:

[0030] S1. Preparation of a carrier: adding 1,6-hexanediol diglycidyl ether and glutaraldehyde to an amino resin in sequence to react to obtain an activated carrier;

[0031] S2. Enzyme immobilization: reacting the enzyme solution composition with the activated carrier obtained in step S1 to obtain the immobilized enzyme, wherein the enzyme solution composition contains α-glucan phosphorylase, glucose phosphomutase, inositol-3-phosphate synthase and inositol monophosphatase.

[0032] The amino resin in step S1 is preferably LX-1000HA resin, and the reaction time of adding 1,6-hexanediol diglycidyl ether for reaction is 12 to 48 hours, such as 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 24 hours, 26 hours, 28 hours, 30 hours, 32 hours, 34 hours, 36 hours, 38 hours, 40 hours, 42 hours, 44 hours, 46 hours, 48 ​​hours or any value therebetween, preferably 24 hours; the reaction time of adding glutaraldehyde for reaction is 5 to 20 hours, such as 5 hours, 10 hours, 15 hours, 20 hours or any value therebetween, preferably 10 hours.

[0033] The mass ratio of 1,6-hexanediol diglycidyl ether to the carrier in step S1 is 1:1-1:2, such as 1:1, 1:1.5, 1:2 or any value therebetween; the mass ratio of glutaraldehyde to the carrier is 1:8-1:12, such as 1:8, 1:9, 1:10, 1:11, 1:12 or any value therebetween.

[0034] In step S1, the amino group of the carrier reacts with the epoxy group in 1,6-hexanediol diglycidyl ether, so that the carrier has a longer arm with an epoxy group. At the same time, the amino group of the carrier reacts with the aldehyde group in glutaraldehyde, so that the carrier has an arm with an aldehyde group. The epoxy arm and the aldehyde arm with different chain lengths react with the amino group in the enzyme to achieve multi-point fixation of the enzyme and increase the stability of the immobilized enzyme. If only glutaraldehyde is used to activate the immobilized enzyme, or only 1,6-hexanediol diglycidyl ether is used to activate the immobilized enzyme, or the activation order of 1,6-hexanediol diglycidyl ether and glutaraldehyde is reversed, the immobilization effect of the obtained enzyme is not as good as the effect of using 1,6-hexanediol diglycidyl ether and glutaraldehyde to participate in the activation in sequence, which is described in the following specific embodiments.

[0035] In step S2, the α-glucan phosphorylase, glucose phosphomutase, inositol-3-phosphate synthase and inositol monophosphatase in the enzyme liquid composition are added in the form of enzyme liquid, and the preparation methods of the enzyme liquid are as follows: centrifuging the fermentation liquid of the α-glucan phosphorylase, glucose phosphomutase, inositol-3-phosphate synthase or inositol monophosphatase to obtain bacterial sludge, and then adding a phosphate aqueous solution and stirring to obtain a homogenous liquid, and heating and centrifuging the homogenous liquid to obtain a supernatant to prepare the enzyme liquid.

[0036] The mass ratio of the α-glucan phosphorylase, phosphoglucomutase, inositol-3-phosphate synthase and inositol monophosphatase to the carrier in step S2 is (1-1.5): (1-1.5): (3-5): (1-1.5): 1, such as 1:1:3:1:1, 1.5:1.5:5:1.5:1 or any value therebetween, preferably (1-1.5): (1-1.5): (4-5): (1-1.5): 1, such as 1:1:4:1:1, 1.5:1.5:5:1.5:1 or any value therebetween.

[0037] After immobilizing four enzymes, namely, α-glucan phosphorylase, phosphoglucomutase, inositol-3-phosphate synthase and inositol monophosphatase, the immobilized enzymes are treated with glycine. The post-treatment of the immobilized enzymes is to place the immobilized enzymes in a glycine solution for reaction. The pH of the glycine solution is 7 to 9, such as 7, 8, 9 or any value therebetween, preferably 8.5. The reaction time is 18 to 30 hours, such as 18 hours, 20 hours, 22 hours, 24 hours, 26 hours, 28 hours, 30 hours or any value therebetween. The reaction temperature is 20 to 28°C, such as 20°C, 22°C, 24°C, 26°C, 28°C or any value therebetween. Preferably, the mass ratio of the glycine to the carrier is 1:1 to 1.5:1, such as 1:1, 1.2:1, 1.5:1 or any value therebetween. The post-treatment of the immobilized enzymes with glycine can make the generated inositol reach a higher concentration in a shorter time.

[0038] The present invention provides an application of the immobilized enzyme or the immobilized enzyme obtained according to the preparation method in catalyzing the reaction of preparing inositol.

[0039] The present invention provides a method for preparing inositol, comprising: subjecting maltodextrin to an enzymatic reaction in the presence of an immobilized enzyme to obtain inositol; preferably, the mass ratio of the maltodextrin to the immobilized enzyme is 1:1-2:1, such as 1:1, 1.5:1, 2:1 or any value therebetween, the reaction temperature of the enzymatic reaction is 65-75°C, such as 65°C, 70°C, 75°C or any value therebetween, and the reaction time is 60-80h, such as 60h, 65h, 70h, 75h, 80h or any value therebetween.

[0040] In the following examples and comparative examples, the resin manufacturer used is Xi'an Lanxiao Technology New Materials Co., Ltd., and the specific models of the resins are LX-1000HA resin, LX-1000EPN resin, LX-1000EA resin and LX-EPHA resin.

[0041] In the following examples and comparative examples, the method for determining inositol refers to GB 1903.42-2020A.3.2 high performance liquid chromatography.

[0042] In the following examples and comparative examples, isoamylase, α-glucan phosphorylase, glucose phosphomutase, inositol-3-phosphate synthase and inositol monophosphatase were prepared with reference to the method in CN109913489A, and then the fermentation broth of isoamylase, α-glucan phosphorylase, glucose phosphomutase, inositol-3-phosphate synthase and inositol monophosphatase was centrifuged to collect the bacterial sludge; 50 g of the bacterial sludge of the above five enzymes was taken, and 150 mL of 50 mM phosphate was added to each (7.8 g of sodium dihydrogen phosphate dihydrate was weighed, and 1 L of purified water was added and stirred to dissolve; 24.0 g of sodium dihydrogen phosphate dodecahydrate was weighed and added to 1.34 L of purified water and stirred to dissolve; and the two were mixed), stirred evenly and homogenized with a high-pressure homogenizer at a homogenization pressure of 800 to 900 bar, homogenized 5 times, and the resulting homogenate was heated at 70 ° C for 30 min, and the supernatant obtained by centrifugation was the enzyme solution of various enzymes.

[0043] Example 1: Preparation of immobilized enzyme

[0044] Take 20g of LX-1000HA resin as a carrier, add 200mL of 50mM phosphate, stir at 25℃ for 6h, filter and obtain the balanced carrier. Add 300mL of 0.2mol / L 1,6-hexanediol diglycidyl ether (0.2mol / L 1,6-hexanediol diglycidyl ether preparation method: weigh 13.8g of 1,6-hexanediol diglycidyl ether, add 300mL of 1-propanol and stir to dissolve) to the balanced carrier and stir at 25℃ for 24h. After filtering, wash the carrier with 50mM phosphate buffer until the water is clear. Add 100mL of 2% glutaraldehyde (2% glutaraldehyde solution preparation: weigh 4g of 50% glutaraldehyde, add 96g of 50mM phosphate buffer, stir evenly) to the carrier after washing, and stir at 25℃ for 10h. After filtration, the carrier was washed with 50 mM phosphate buffer until the water was clear.

[0045] Take 20 mL of α-glucan phosphorylase solution, 20 mL of glucose phosphomutase solution, 80 mL of inositol-3-phosphate synthase solution, and 20 mL of inositol monophosphatase solution, mix them evenly, add 20 g of the above-mentioned activated carrier, stir at 25°C for 24 h, filter and collect the immobilized enzyme after immobilization, wash it 3 times with purified water, and drain it. The obtained immobilized enzyme is placed in 100 mL of 3 mol / L pH=8.5 glycine solution (3 mol / L glycine solution preparation: weigh 22.5 g of glycine, add purified water to dissolve, and then adjust the volume to 100 mL, and adjust the pH to 8.5 with 10% sodium hydroxide), stir at 25°C for 24 h, wash it 3 times with purified water, and drain it to obtain the immobilized enzyme. The obtained immobilized enzyme was used to prepare inositol according to the following steps: 30 g of maltodextrin was weighed, 70 mL of purified water was added, 0.68 g of potassium dihydrogen phosphate was added, 0.06 g of magnesium sulfate was added, and the mixture was heated at 80°C, pH was adjusted to 5.0-5.5 with 10% potassium hydroxide, 5 mL of isoamylase solution was added, and the mixture was reacted at 80°C for 3 h. The temperature of the reaction solution was lowered to 70°C, pH was adjusted to 7.0-7.5 with 10% potassium hydroxide, 20 g of immobilized enzyme was added to the reaction solution, the reaction temperature was controlled to 70°C, pH was adjusted to 7.0-7.5, the reaction was continued for 70 h, and the inositol content was determined by sampling. The inositol concentration was 219 g / L, and the conversion rate was 73%.

[0046] Example 2: Effect of different carriers on immobilization effect

[0047] LX-1000EPN resin, LX-1000EA resin and LX-EPHA resin were used as different carriers, and the rest was carried out with reference to Example 1 to obtain immobilized enzymes. The concentrations of inositol prepared by the obtained immobilized enzymes were 151 g / L, 142 g / L and 130 g / L, respectively, and the conversion rates were 50%, 47% and 43%, respectively. It can be seen from Example 1 and this example that among the four different carriers, LX-1000HA resin has a better immobilization effect as a carrier.

[0048] Examples 3 to 6: Effects of different mass ratios of four thermostable enzymes on the immobilization effect

[0049] Different mass ratios of enzyme solutions of four thermostable enzymes are shown in Table 1, and other experimental conditions are the same as those in Example 1 to prepare immobilized enzymes, which are then further used to prepare inositol.

[0050] Table 1 Effect of different mass ratios of four thermostable enzymes on the immobilization effect

[0051] Thermostable enzyme Example 3 Example 4 Example 5 Example 6 α-Glucan phosphorylase 20mL 20mL 10mL 30mL Phosphoglucomutase 20mL 20mL 10mL 30mL Inositol-3-phosphate synthase 60mL 100mL 80mL 80mL Inositol monophosphatase 20mL 20mL 10mL 30mL Preparation of inositol concentration 156g / L 225g / L 132g / L 216g / L Inositol conversion rate 52％ 75％ 44％ 72％

[0052] As shown in Table 1, the performance of Example 4 and Example 6 in preparing inositol concentration and inositol conversion rate is better than that of Example 3 and Example 5. When the immobilization conditions are that the mass ratio of α-glucan phosphorylase, phosphoglucomutase, inositol-3-phosphate synthase and inositol monophosphatase to the carrier is preferably (1-1.5): (1-1.5): (4-5): (1-1.5): 1, the carrier has excellent immobilization effect on the four heat-resistant enzymes. Among them, the inositol concentration prepared in Example 4 reaches 225g / L, and the conversion rate is 75%.

[0053] Comparative Example 1: 1,6-Hexanediol diglycidyl ether does not participate in the activation of immobilized enzyme

[0054] The same reaction materials and reaction conditions as in Example 1 were used, except that 1,6-hexanediol diglycidyl ether was not added, and activation of the carrier by glutaraldehyde was retained to obtain an immobilized enzyme. The inositol content was 132.8 g / L and the conversion rate was 44.3%.

[0055] Comparative Example 2: Glutaraldehyde does not participate in the activation of immobilized enzymes

[0056] The same reaction materials and reaction conditions as in Example 1 were used, except that glutaraldehyde was not added and 1,6-hexanediol diglycidyl ether was retained to activate the carrier, thereby obtaining an immobilized enzyme. The inositol content was 163.8 g / L and the conversion rate was 54.6%.

[0057] Comparative Example 3: Reversing the activation order of immobilized enzymes

[0058] The same reaction materials and reaction conditions as in Example 1 were used, except that the activation order of 1,6-hexanediol diglycidyl ether and glutaraldehyde was opposite to that in Example 1, i.e., glutaraldehyde was used for activation first, and then 1,6-hexanediol diglycidyl ether was used for activation to obtain the immobilized enzyme. The inositol content was 150 g / L and the conversion rate was 50%.

[0059] Based on the results of Example 1, Comparative Example 1, Comparative Example 2 and Comparative Example 3, it can be seen that the immobilization effect of the obtained enzyme is not as good as the effect of using 1,6-hexanediol diglycidyl ether and glutaraldehyde to participate in the activation of the immobilized enzyme only by using glutaraldehyde, or only by using 1,6-hexanediol diglycidyl ether to participate in the activation of the immobilized enzyme, or reversing the activation order of 1,6-hexanediol diglycidyl ether and glutaraldehyde in sequence. Using cross-linking agents of different chain lengths to activate the amino resin in sequence is conducive to achieving multi-point fixation of the enzyme and increasing the stability of the immobilized enzyme.

[0060] Example 7: Reuse of immobilized enzyme

[0061] Using the same reaction materials and reaction conditions as in Example 1, the immobilized enzyme was repeatedly fed 10 times, and the resulting inositol contents were 216g / L, 219g / L, 213g / L, 210g / L, 213g / L, 212g / L, 195g / L, 189g / L, 174g / L and 165g / L, respectively, and the conversion rates were 72%, 73%, 71%, 70%, 71%, 71%, 65%, 63%, 58% and 55%, respectively. The conversion rate was 70% to 73% after the first 6 reuses, and the conversion rate was reduced to 55% after 10 reuses.

[0062] Comparative Example 4: Immobilized enzyme without glycine post-treatment

[0063] The reaction materials and reaction conditions identical with those in Example 1 were adopted, except that the immobilized enzyme was not subjected to glycine post-treatment and then used for the synthesis of inositol. The resulting inositol contents were 180 g / L, 162 g / L, 120 g / L, 85 g / L, 65 g / L, 50 g / L, 42 g / L, 40 g / L, 35 g / L and 33 g / L, respectively, and the conversion rates were 60%, 54%, 40%, 28%, 22%, 17%, 14%, 13%, 12% and 11%, respectively. The conversion rate was 60% after the first reuse, and the conversion rate decreased gradually with the increase of the number of reuses, and the conversion rate was reduced to 11% after 10 reuses.

[0064] Based on the results of Example 7 and Comparative Example 4, it can be seen that the immobilized enzyme is not post-treated with glycine, and the effect of the obtained immobilized enzyme is poor. The main reason is that without post-treatment with glycine, the excess aldehyde and epoxy groups of the carrier may react with the active site of the enzyme, resulting in reduced enzyme activity. In Example 7, the immobilized enzyme was reused 6 times and its conversion rate was still greater than 70%. Post-treatment of the immobilized enzyme with glycine is beneficial to reducing the cost of inositol production.

[0065] The above describes the specific implementation of the present application, but the protection scope of the present application is not limited thereto. Any technician familiar with the technical field can easily think of changes or substitutions within the technical scope disclosed in the present application, which should be included in the protection scope of the present application. Therefore, the protection scope of the present application should be based on the protection scope of the claims.

Claims

1. An immobilized enzyme, characterized in that The amino resin is used as a carrier, and the carrier is immobilized with enzymes, which include alpha-glucan phosphorylase, phosphoglucomutase, inositol-3-phosphate synthase and inositol monophosphatase.

2. The immobilized enzyme according to claim 1, characterized in that The amino resin is LX-1000HA resin.

3. The immobilized enzyme according to claim 1, characterized in that The mass ratio of the α-glucan phosphorylase, phosphoglucomutase, inositol-3-phosphate synthase and inositol monophosphatase to the carrier is (1-1.5): (1-1.5): (3-5): (1-1.5): 1, preferably (1-1.5): (1-1.5): (4-5): (1-1.5):

1.

4. The method for preparing an immobilized enzyme according to any one of claims 1 to 3, characterized in that: The following steps are involved: S1. Activation of the carrier: adding 1,6-hexanediol diglycidyl ether and glutaraldehyde to the amino resin in sequence to react to obtain an activated carrier; S2. Enzyme immobilization: reacting the enzyme solution composition with the activated carrier obtained in step S1 to obtain the immobilized enzyme, wherein the enzyme solution composition contains α-glucan phosphorylase, glucose phosphomutase, inositol-3-phosphate synthase and inositol monophosphatase.

5. The preparation method according to claim 4, characterized in that: The reaction time of adding 1,6-hexanediol diglycidyl ether for reaction in step S1 is 12 to 48 hours, preferably 24 hours; the reaction time of adding glutaraldehyde for reaction is 5 to 20 hours, preferably 10 hours.

6. The preparation method according to claim 4, characterized in that: The mass ratio of the 1,6-hexanediol diglycidyl ether to the carrier is 1:1 to 1:2; the mass ratio of the glutaraldehyde to the carrier is 1:8 to 1:

12.

7. The preparation method according to claim 4, characterized in that: The method further comprises placing the immobilized enzyme obtained in step S2 in a glycine solution for reaction.

8. The preparation method according to claim 7, characterized in that: The pH of the glycine solution is 7 to 9, preferably 8.5; the reaction time is 18 to 30 hours, and the reaction temperature is 20 to 28° C.; preferably, the mass ratio of the glycine to the carrier is 1:1 to 1.5:

1.

9. Use of the immobilized enzyme according to any one of claims 1 to 3 or the immobilized enzyme obtained according to the preparation method according to any one of claims 4 to 8 in catalyzing the reaction of preparing inositol.

10. A method for preparing inositol, characterized in that: The following steps are involved: In the presence of the immobilized enzyme according to any one of claims 1 to 3 or the immobilized enzyme obtained according to the preparation method according to any one of claims 4 to 8, maltodextrin is subjected to an enzymatic reaction to obtain inositol; preferably, the mass ratio of the maltodextrin to the immobilized enzyme is 1:1 to 2:1, the reaction temperature of the enzymatic reaction is 65 to 75°C, and the reaction time is 60 to 80 hours.

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