Multi-enzyme system, preparation method thereof and application of multi-enzyme system in preparation of inositol

Abstract

The invention discloses a multienzyme system, a preparation method thereof and application of the multienzyme system in inositol preparation. According to the preparation method of the inositol disclosed by the invention, maltodextrin is converted into the inositol by using a multi-enzyme system; the multi-enzyme system is prepared from alpha-glucan phosphorylase, glucose phosphate mutase, inositol-1-phosphate synthetase and phytase. The multi-enzyme system provided by the invention is used for preparing the inositol, and has the advantages of low cost of raw materials and enzymes, short reaction time, few byproducts, high product purity, high yield and high yield.

Description

Technical Field

[0001] The enzyme catalysis production field of the present invention relates to a multi-enzyme system and a preparation method thereof and application in the preparation of inositol. Background Art

[0002] Inositol, also known as cyclohexanehexol, has a molecular formula of C6H 12 O6 is a white crystal or crystalline powder, odorless, slightly sweet, widely distributed in animals and plants, and is an essential substance for the growth of humans, animals and microorganisms. Inositol has a wide range of uses: in the feed industry, as a biological promoter, it can promote livestock growth and prevent hair loss; in the food industry, inositol is often used as a nutritional enhancer to prevent fat from depositing in the human body, especially in the cardiovascular system; in the medical field, inositol can be used as a drug to assist in the treatment of mental and neurological diseases, vitamin deficiency, vascular sclerosis, diabetes, cirrhosis and other diseases.

[0003]

[0004] The traditional method of producing inositol is to use calcium phytate obtained from rice bran, soybean meal, etc. as raw material, and further hydrolyze and purify it to obtain inositol. However, due to the many disadvantages of this production method, such as expensive raw materials, serious phosphorus pollution, complex separation of raw materials and products, and low raw material utilization, it leads to relatively high prices and limited supply. At present, the emerging method of producing inositol is to use phytase to hydrolyze phytic acid into inositol through a series of complex processes. Although this method reduces the pollution generated in the production process, it still needs phytic acid as raw material and does not reduce production costs.

[0005] Based on the research on the metabolic pathway of inositol, the microbial fermentation method has also been developed to a certain extent. Inositol is produced by fermentation of Candida albicans, Saccharomyces cerevisiae, and Escherichia coli selected from nature or genetically engineered, and then high-purity inositol is obtained by processing the fermentation liquid and concentrating and crystallizing. The production cost of this method is reduced, but it is difficult to increase the production of inositol due to the competition of other metabolic pathways for carbon flux.

[0006] Compared with the above methods, the synthesis of inositol using easily available raw materials such as starch, cellulose, sucrose, and glucose through an in vitro multi-enzyme catalytic system has more advantages in industrial production due to its environmental friendliness, fast conversion rate, and mild conversion conditions.

[0007] Patent CN105925643A discloses a method for preparing inositol by enzymatic catalysis, using starch as substrate and using α-glucan phosphorylase, hexose phosphomutase, inositol phosphate synthase and phosphoinositide phosphatase from different thermophilic microorganisms to obtain inositol in a one-step reaction. The reaction is carried out at 70-90℃ for 72h, with a maximum conversion rate of 62%. After subsequent purification, the purity of inositol can reach more than 95%.

[0008] The above methods have a long reaction time, and the yield of inositol still has room for improvement. Therefore, there is still a need to develop a more efficient method for preparing inositol by enzyme catalysis. Summary of the invention

[0009] In view of the problems of high raw material cost, low inositol yield or long reaction time in the existing method for preparing inositol, the main purpose of the present invention is to provide a multi-enzyme system and a preparation method thereof and application in the preparation of inositol, which adopts heat-resistant isoamylase, heat-resistant α-glucan phosphorylase, heat-resistant glucose phosphomutase, heat-resistant inositol-1-phosphate synthase and heat-resistant phytase to catalyze high-concentration maltodextrin (100g / L-180g / L) to prepare high-concentration inositol.

[0010] The present invention uses maltodextrin as a raw material to produce inositol, instead of using phytic acid (inositol hexaphosphate) as a raw material to produce inositol. Maltodextrin is made from corn or rice as a raw material, and is hydrolyzed and liquefied by an enzyme method (such as high-temperature resistant α-amylase), decolorized, filtered, ion exchanged, vacuum concentrated and spray dried. It has a wide source and a low raw material cost. Therefore, the cost of preparing inositol using maltodextrin as a raw material is much lower than the cost of generating inositol by enzymatic hydrolysis using phytic acid as a raw material.

[0011] The first aspect of the present invention provides a method for preparing inositol, which uses a multi-enzyme system to convert maltodextrin into inositol; the multi-enzyme system comprises α-glucan phosphorylase, phosphoglucomutase, inositol-1-phosphate synthase and phytase.

[0012] In some preferred embodiments, the amino acid sequence of the phytase is selected from any one or more of SEQ ID NOs: 9, 11, 13 and 15; and / or,

[0013] The heat-resistant temperature range of the phytase is 55°C to 75°C.

[0014] Preferably, the nucleotide sequence encoding the phytase is selected from any one or more of SEQ ID NOs: 10, 12, 14 and 16.

[0015] In the present invention, "plurality" means two or more.

[0016] In some more preferred embodiments, the amino acid sequence of the α-glucan phosphorylase is shown in SEQ ID NO: 1; and / or, the amino acid sequence of the glucose phosphomutase is shown in SEQ ID NO: 3; and / or, the amino acid sequence of the inositol-1-phosphate synthase is shown in SEQ ID NO: 5; and / or,

[0017] The heat resistant temperature range of any one or more of the α-glucan phosphorylase, the phosphoglucomutase and the inositol-1-phosphate synthase is 55° C. to 75° C.

[0018] Preferably, the nucleotide sequence encoding the α-glucan phosphorylase is shown as SEQ ID NO: 2, and / or the nucleotide sequence encoding the phosphoglucomutase is shown as SEQ ID NO: 4, and / or the nucleotide sequence encoding the inositol-1-phosphate synthase is shown as SEQ ID NO: 6.

[0019] In some preferred embodiments, the multi-enzyme system further comprises isoamylase.

[0020] Preferably, the amino acid sequence of the isoamylase is as shown in SEQ ID NO: 7; and / or the heat-resistant temperature range of the isoamylase is 55° C. to 75° C.;

[0021] More preferably, the nucleotide sequence encoding the isoamylase is shown as SEQ ID NO:8.

[0022] In other preferred embodiments, in the reaction system consisting of at least the multi-enzyme system and the maltodextrin, the initial concentration of the maltodextrin is 10 to 190 g / L, preferably 100 to 180 g / L.

[0023] In the present invention, "initial concentration" or "initial mass volume concentration" (unit: g / L) refers to the components in the reaction system, such as maltodextrin or Mg 2+ The initial concentration when the component is added to the reaction system, that is, the concentration of the component in the reaction system before the reaction starts (reaction time is 0).

[0024] In other preferred embodiments, the conversion is carried out at a temperature of 58°C to 65°C, preferably 60°C to 63°C; for example, an oil bath of 65°C to 75°C or a metal bath of 60°C to 65°C is used to maintain the temperature.

[0025] In the present invention, the temperature of the conversion refers to the actual measured temperature of the conversion reaction liquid. The metal bath is a metal in direct contact with the reaction liquid, and the metal transfers heat very quickly. The temperature of the metal bath is approximately equal to the temperature of the reaction liquid; the oil bath will transfer heat to the oil medium through the heating plate, and then transfer heat to the reaction liquid. Therefore, the set temperature of the oil bath will be higher.

[0026] And / or, the conversion is carried out in a phosphate buffer having a pH value of 6.9 to 7.1; preferably, the phosphate buffer is a potassium phosphate buffer; more preferably, the pH value of the potassium phosphate buffer is 7.0.

[0027] And / or, adding Mg before or during the conversion 2+ Preferably, the Mg 2+ The initial concentration is 7 mM to 9 mM, for example 8 mM.

[0028] And / or, the conversion is carried out under shaking or stirring; preferably, the stirring is magnetic stirring, and / or, the stirring speed is 300 rpm to 600 rpm.

[0029] And / or, the duration of the conversion is 15h to 36h, preferably 28h to 30h.

[0030] In the present invention, theoretically, after the conversion exceeds 30 hours, if the reaction is continued, the content of inositol will increase, but the increase is very small, so the actual reaction end point is about 3 days.

[0031] The second aspect of the present invention provides a method for preparing a multi-enzyme system, which comprises the following steps: mixing α-glucan phosphorylase, phosphoglucomutase, inositol-1-phosphate synthase and phytase to obtain the multi-enzyme system.

[0032] In some preferred embodiments, the amino acid sequence of the α-glucan phosphorylase is as shown in SEQ ID NO: 1; and / or the amino acid sequence of the glucose phosphomutase is as shown in SEQ ID NO: 3; and / or the amino acid sequence of the inositol-1-phosphate synthase is as shown in SEQ ID NO: 5; and / or the amino acid sequence of the phytase is selected from any one or more of SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13 and SEQ ID NO: 15.

[0033] In some preferred embodiments, the nucleotide sequence encoding the α-glucan phosphorylase is shown as SEQ ID NO: 2; and / or, the nucleotide sequence encoding the phosphoglucomutase is shown as SEQ ID NO: 4; and / or, the nucleotide sequence encoding the inositol-1-phosphate synthase is shown as SEQ ID NO: 6; and / or, the nucleotide sequence encoding the phytase is selected from any one or more of SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14 and SEQ ID NO: 16.

[0034] In some preferred embodiments, the α-glucan phosphorylase, the phosphoglucomutase, the inositol-1-phosphate synthase and the phytase exist alone or together in the form of whole-cell enzyme, homogenized enzyme solution, crude enzyme solution or solid enzyme powder.

[0035] In the present invention, the whole-cell enzyme is produced by cells (e.g., prokaryotic cells) containing genes encoding any one or more enzymes in the multi-enzyme system; whole-cell catalysis refers to the use of complete biological organisms (i.e., cells, tissues, or even individuals) for catalytic transformation, and its essence is to use enzymes in cells for catalysis. This method is a biocatalytic technology between fermentation and free enzyme catalysis.

[0036] In the present invention, the homogenized enzyme solution is an enzyme solution obtained by resuspending wet bacteria with a resuspending solution and breaking the cells, without heating and / or heat treatment; the wet bacteria are the precipitate obtained after solid-liquid separation (i.e., discarding the supernatant after centrifugation and taking the precipitate) of the culture solution of the prokaryotic cells.

[0037] In the present invention, cells can be disrupted by any conventional method in the art, including but not limited to: high pressure disruption, ultrasonic disruption, osmotic shock disruption, repeated freezing and thawing, lysozyme treatment or cell lysis solution treatment.

[0038] In the present invention, the crude enzyme solution is the enzyme solution obtained after the homogenous enzyme solution is removed from impurities (i.e., the supernatant is taken after centrifugation and the precipitate is discarded) and heated. In the present invention, "heating" specifically refers to the process of heating the homogenous enzyme solution of a certain enzyme to obtain its crude enzyme solution, that is, it only refers to the process from "homogeneous enzyme solution" to "crude enzyme solution". The heating includes: before the homogenous enzyme solutions of various enzymes are mixed into a multi-enzyme system, the homogenous enzyme solution of the enzyme is heated; preferably, before and / or after the heating, the precipitate of the homogenous enzyme solution of the enzyme is discarded to obtain the supernatant; the precipitate is preferably produced by centrifugation or standing.

[0039] In the present invention, if some components of the multi-enzyme system are homogenized enzyme liquids and some components are crude enzyme liquids, then the homogenized enzyme liquids and the crude enzyme liquids are subjected to a heat treatment process after being mixed, that is, the components of the crude enzyme liquids in the final multi-enzyme system are equivalent to undergoing a "heating" and a "heat treatment" process. The "heat treatment" in the present invention specifically refers to the heat treatment of the entire multi-enzyme system after various enzymes are mixed together, including the process of heat treatment again after some components of the entire multi-enzyme system are homogenized enzyme liquids and other components are crude enzyme liquids (which have been heated by homogenized enzyme liquids).

[0040] In the present invention, the solid enzyme powder is a powder obtained by drying and pulverizing the crude enzyme solution.

[0041] In some preferred embodiments, any one or more of the α-glucan phosphorylase, the glucose phosphomutase, the inositol 1-phosphate synthase and the phytase are present in the form of a homogenized enzyme solution, and the ratio of the added mass of wet bacteria to the added volume of the resuspension in the homogenized enzyme solution is 1 g:(5-10) mL.

[0042] In some preferred embodiments, the homogenized enzyme solution is heated before mixing, and the heating temperature is 58°C to 65°C; preferably, 60°C to 63°C; and / or, the heating time is 30min to 60min, preferably 30min to 35min. In the present invention, the heating temperature refers to the actual measured temperature, not the temperature displayed on the instrument. The purpose of this step is to make the homogenized enzyme solution into a crude enzyme solution.

[0043] In some more preferred embodiments, the resuspension solution is water, a buffer (eg, phosphate buffer) or a culture medium (eg, LB basal medium or TB basal medium).

[0044] In some more preferred embodiments, the phosphate buffer is a potassium phosphate buffer; and / or the pH value of the phosphate buffer is 6.9 to 7.1, preferably 7.0.

[0045] In some preferred embodiments, during or after mixing, heat treatment is performed; preferably, the temperature of the heat treatment is 58°C to 65°C; preferably, 60°C to 63°C; and / or, the time of the heat treatment is 30min to 60min, preferably 30min to 35min. In the present invention, the temperature of the heat treatment refers to the actual measured temperature, not the temperature displayed on the instrument. The purpose of this step is to heat treat the entire multi-enzyme system.

[0046] In some preferred embodiments, in the multi-enzyme system, the addition amounts of the α-glucan phosphorylase, the phosphoglucomutase, the inositol-1-phosphate synthase and the phytase are calculated according to the addition mass of the wet cells as follows:

[0047] The added mass ratio of the wet bacteria capable of producing the α-glucan phosphorylase to the wet bacteria capable of producing the phosphoglucomutase is 1:(0.15-0.4); and / or,

[0048] The added mass ratio of the wet bacteria capable of producing the α-glucan phosphorylase to the wet bacteria capable of producing the inositol-1-phosphate synthase is 1:(2-6); and / or,

[0049] The mass ratio of the wet bacteria that can produce the α-glucan phosphorylase to the wet bacteria that can produce the phytase is 1:(0.75-7.5).

[0050] In some preferred embodiments, the method further comprises: adding isoamylase before, during or after the mixing.

[0051] In some preferred embodiments, the amino acid sequence of the isoamylase is as shown in SEQ ID NO: 7; and / or, the isoamylase exists in the form of whole-cell enzyme, homogenized enzyme solution, crude enzyme solution or solid enzyme powder.

[0052] In some more preferred embodiments, the nucleotide sequence encoding the isoamylase is shown in SEQ ID NO: 8; and / or, under the condition that the isoamylase is present in the form of the homogenized enzyme solution, it is heated before the mixing, and / or, it is heat-treated during or after the mixing; and / or

[0053] In the multi-enzyme system, the addition amount of the α-glucan phosphorylase and the isoamylase is calculated according to the added mass of the wet bacteria as follows: the mass ratio of the wet bacteria that can produce the α-glucan phosphorylase to the wet bacteria that can produce the isoamylase is 1:(0.25-0.85).

[0054] Further preferably, the temperature of the heating and / or heat treatment is 58°C to 65°C; preferably, 60°C to 63°C; and / or,

[0055] The heating and / or heat treatment time is 30 min to 60 min, preferably 30 min to 35 min.

[0056] The third aspect of the present invention provides a multi-enzyme system, which comprises α-glucan phosphorylase, phosphoglucomutase, inositol-1-phosphate synthase and phytase.

[0057] In some preferred embodiments, the α-glucan phosphorylase, the phosphoglucomutase, the inositol-1-phosphate synthase and the phytase are present alone or together in the form of whole-cell enzyme, homogenized enzyme solution, crude enzyme solution or solid enzyme powder; and / or

[0058] The multi-enzyme system further comprises isoamylase; and / or

[0059] The amino acid sequence of the α-glucan phosphorylase is shown in SEQ ID NO: 1; and / or,

[0060] The amino acid sequence of the phosphoglucomutase is shown in SEQ ID NO: 3; and / or,

[0061] The amino acid sequence of the inositol-1-phosphate synthase is shown in SEQ ID NO: 5; and / or,

[0062] The amino acid sequence of the phytase is selected from any one or more of SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13 and SEQ ID NO:15.

[0063] In some more preferred embodiments, the isoamylase is present in the form of whole cell enzyme, homogenized enzyme solution, crude enzyme solution or solid enzyme powder; and / or,

[0064] The amino acid sequence of the isoamylase is shown in SEQ ID NO: 7, preferably, the nucleotide sequence encoding the isoamylase is shown in SEQ ID NO: 8; and / or,

[0065] The nucleotide sequence encoding the α-glucan phosphorylase is shown in SEQ ID NO: 2; and / or,

[0066] The nucleotide sequence encoding the phosphoglucomutase is shown in SEQ ID NO:4; and / or,

[0067] The nucleotide sequence encoding the inositol-1-phosphate synthase is shown in SEQ ID NO: 6; and / or,

[0068] The nucleotide sequence encoding the phytase is selected from any one or more of SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14 and SEQ ID NO:16.

[0069] The fourth aspect of the present invention provides an application of the multi-enzyme system as described in the third aspect of the present invention, or the multi-enzyme system obtained by the preparation method described in the second aspect of the present invention, in catalyzing the production of inositol using maltodextrin as a substrate.

[0070] In a fifth aspect, the present invention provides a use of a phytase in catalyzing the production of inositol from inositol-1-phosphate, wherein the amino acid sequence of the phytase is selected from any one or more of SEQ ID NOs: 9, 11, 13 and 15.

[0071] Preferably, the inositol-1-phosphate is obtained by catalyzing α-glucan phosphorylase, phosphoglucomutase and inositol-1-phosphate synthase in sequence (stepwise method) or together (one-pot method) with maltodextrin as substrate; or, the inositol-1-phosphate is obtained by catalyzing α-glucan phosphorylase, phosphoglucomutase and inositol-1-phosphate synthase in sequence or together with maltodextrin as substrate.

[0072] More preferably, the amino acid sequence of the α-glucan phosphorylase is as shown in SEQ ID NO: 1; and / or,

[0073] The amino acid sequence of the phosphoglucomutase is shown in SEQ ID NO: 3; and / or,

[0074] The amino acid sequence of the inositol-1-phosphate synthase is shown in SEQ ID NO: 5; and / or,

[0075] The amino acid sequence of the isoamylase is shown in SEQ ID NO:7.

[0076] Further more preferably, the nucleotide sequence encoding the α-glucan phosphorylase is as shown in SEQ ID NO: 2; and / or,

[0077] The nucleotide sequence encoding the phosphoglucomutase is shown in SEQ ID NO:4; and / or,

[0078] The nucleotide sequence encoding the inositol-1-phosphate synthase is shown in SEQ ID NO: 6; and / or,

[0079] The nucleotide sequence encoding the isoamylase is shown in SEQ ID NO: 8; and / or,

[0080] The nucleotide sequence encoding the phytase is selected from any one or more of SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14 and SEQ ID NO:16.

[0081] The sixth aspect of the present invention provides an enzyme-catalyzed reaction system, the enzyme-catalyzed reaction system comprising: maltodextrin, α-glucan phosphorylase, glucose phosphomutase, inositol-1-phosphate synthase and phytase; and a buffer and Mg 2+ One or two of; preferably the buffer is a phosphate buffer such as a potassium phosphate buffer; more preferably the pH of the buffer is 6.9 to 7.1, such as 7.0.

[0082] In the present invention, the enzyme catalytic reaction system at least contains maltodextrin, α-glucan phosphorylase, phosphoglucomutase, inositol-1-phosphate synthase and phytase. The enzyme catalytic reaction system preferably contains maltodextrin, buffer, α-glucan phosphorylase, phosphoglucomutase, inositol-1-phosphate synthase and phytase.

[0083] Preferably, the enzyme catalyzed reaction system further comprises inositol and / or isoamylase; and / or,

[0084] The amino acid sequence of the α-glucan phosphorylase is shown in SEQ ID NO: 1; and / or,

[0085] The amino acid sequence of the phosphoglucomutase is shown in SEQ ID NO: 3; and / or,

[0086] The amino acid sequence of the inositol-1-phosphate synthase is shown in SEQ ID NO: 5; and / or,

[0087] The amino acid sequence of the phytase is selected from any one or more of SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13 and SEQ ID NO: 15; and / or,

[0088] The initial concentration of the maltodextrin in the enzyme-catalyzed reaction system is 10 to 190 g / L.

[0089] More preferably, the nucleotide sequence encoding the α-glucan phosphorylase is as shown in SEQ ID NO: 2; and / or,

[0090] The nucleotide sequence encoding the phosphoglucomutase is shown in SEQ ID NO:4; and / or,

[0091] The nucleotide sequence encoding the inositol-1-phosphate synthase is shown in SEQ ID NO: 6; and / or,

[0092] The nucleotide sequence encoding the phytase is selected from any one or more of SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14 and SEQ ID NO: 16; and / or,

[0093] The amino acid sequence of the isoamylase is shown in SEQ ID NO: 7; and / or,

[0094] The initial concentration of the maltodextrin in the enzyme-catalyzed reaction system is 100-180 g / L.

[0095] Further more preferably, the nucleotide sequence encoding the isoamylase is shown as SEQ ID NO:8.

[0096] A seventh aspect of the present invention provides a reaction end product system, the reaction end product system comprising:

[0097] Maltodextrin;

[0098] α-glucan phosphorylase, which can hydrolyze the α-1,4 glycosidic bonds in the maltodextrin to release glucose-1-phosphate;

[0099] Phosphoglucomutase, capable of converting the glucose-1-phosphate into glucose-6-phosphate;

[0100] Inositol-1-phosphate synthase, capable of converting the glucose-6-phosphate into inositol-1-phosphate;

[0101] a phytase capable of converting the inositol-1-phosphate into inositol; and

[0102] Inositol, wherein the mass volume concentration (g / L) of the inositol in the final reaction product system accounts for more than 79% of the initial mass volume concentration (g / L) of maltodextrin.

[0103] The "yield" defined in the present invention refers to the ratio of the mass volume concentration (g / L) of the product in the final product system of the reaction to the initial mass volume concentration (g / L) of the substrate.

[0104] Preferably, the mass volume concentration (g / L) of the inositol in the final reaction product system accounts for more than 96% of the initial mass volume concentration (g / L) of the maltodextrin; and / or,

[0105] The amino acid sequence of the α-glucan phosphorylase is shown in SEQ ID NO: 1; and / or,

[0106] The amino acid sequence of the phosphoglucomutase is shown in SEQ ID NO: 3; and / or,

[0107] The amino acid sequence of the inositol-1-phosphate synthase is shown in SEQ ID NO: 5; and / or,

[0108] The amino acid sequence of the phytase is selected from any one or more of SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13 and SEQ ID NO: 15; and / or,

[0109] The reaction final product system also includes: isoamylase, which can hydrolyze the α-1,6 glycosidic bonds of the branching points of the maltodextrin.

[0110] More preferably, the mass volume concentration (g / L) of the inositol in the final reaction product system accounts for more than 97% of the initial mass volume concentration (g / L) of the maltodextrin; and / or,

[0111] The nucleotide sequence encoding the α-glucan phosphorylase is shown in SEQ ID NO: 2; and / or,

[0112] The nucleotide sequence encoding the phosphoglucomutase is shown in SEQ ID NO:4; and / or,

[0113] The nucleotide sequence encoding the inositol-1-phosphate synthase is shown in SEQ ID NO: 6; and / or,

[0114] The nucleotide sequence encoding the phytase is selected from any one or more of SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14 and SEQ ID NO: 16; and / or,

[0115] The amino acid sequence of the isoamylase is shown in SEQ ID NO:7.

[0116] Further more preferably, the nucleotide sequence encoding the isoamylase is shown as SEQ ID NO:8.

[0117] On the basis of being in accordance with the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0118] The reagents and raw materials used in the present invention are commercially available.

[0119] The positive and progressive effects of the present invention include any one or more of the following:

[0120] The present invention adopts a multienzyme system with a suitable temperature of 58° C. to 65° C., which can catalyze maltodextrin to produce inositol at a temperature of 58° C. to 65° C. Therefore, the multienzyme system of the present invention can carry out a long-term catalytic reaction at the reaction temperature without deactivation and can maintain a high enzyme activity within the temperature range, so there is no need to add enzyme liquid during the reaction process, which reduces the cost of using enzymes, and is conducive to maintaining the catalytic activity of the enzyme during the reaction process and shortening the reaction time.

[0121] The reaction temperature of the present invention when producing inositol is 58° C. to 65° C. The multi-enzyme system has a high catalytic activity at the reaction temperature and has substantially no catalytic activity at a lower temperature, while the heat-labile enzyme loses its catalytic activity at the reaction temperature. Therefore, the present invention is not prone to side reactions, which improves the purity of inositol in the final reaction solution and also improves the yield of inositol. In addition, at the reaction temperature, the multi-enzyme system catalyzes the reaction rate of producing inositol from maltodextrin at a faster rate. Therefore, the present invention has a shorter reaction time and a faster reaction rate.

[0122] The multi-enzyme system provided by the present invention is used to prepare inositol, which has the advantages of low raw material and enzyme costs, high inositol yield, short reaction time, few by-products, high product purity and high yield. BRIEF DESCRIPTION OF THE DRAWINGS

[0123] Figure 1 This is the SDS-PAGE detection result of the related enzymes required for the in vitro production of inositol-1-phosphate expressed by Escherichia coli; Figure 1 In a, columns 2 and 3 are α-glucan phosphorylase (αGP), and columns 4 and 5 are phosphoglucomutase (PGM); Figure 1 In b, columns 2 and 3 are inositol 1-phosphate synthase (IPS), and columns 4 and 5 are isoamylase (IA). DETAILED DESCRIPTION

[0124] In order to further illustrate the technical means and effects of the present invention, the following is a detailed description of the preferred embodiments of the present invention in conjunction with the accompanying drawings, but the present invention is not limited to the scope of the embodiments. The experimental methods without specific conditions in the following embodiments are selected according to conventional methods and conditions, or according to the product instructions.

[0125] Unless otherwise specified, the various materials and reagents used in the examples of the present invention are commonly used materials and reagents in the art and can be obtained through conventional commercial channels.

[0126] The biological materials and reagents used in the examples are shown in Table 1 below:

[0127] Table 1 Biological materials and reagents

[0128]

[0129]

[0130] TB medium formula: tryptone 10 g / L; yeast extract 18 g / L; glycerol 4 mL / L; dipotassium hydrogen phosphate 16.43 g / L; potassium dihydrogen phosphate 2.31 g / L.

[0131] LB medium formula: peptone 10g / L, yeast powder 5g / L, NaCl 10g / L.

[0132] The high performance liquid chromatography (HPLC) detection method is:

[0133] A high performance liquid chromatograph equipped with a differential detector was used, with a chromatographic column: Sepax HP-Amide (250×4.6mm, 5μm); mobile phase: acetonitrile: water = 68:32; flow rate: 1.4mL / min; column temperature: 35°C; injection volume: 10μL. Inositol reference substance is required for HPLC detection, and its preparation method is: accurately weigh 25mg of inositol reference substance into a 5ml volumetric flask, add water to dissolve and dilute to the scale, shake well, and use it as inositol reference substance solution. In the chromatogram, the retention time of inositol reference substance is 6.873min.

[0134] Example 1 Preparation of homogenized enzyme solution of related enzymes required for in vitro production of inositol-1-phosphate

[0135] The related enzymes required for the in vitro production of inositol-1-phosphate include isoamylase (IA), α-glucan phosphorylase (αGP), phosphoglucomutase (PGM), and inositol-1-phosphate synthase (IPS). Among them, IA is used to hydrolyze the α-1,6 glycosidic bonds at the branching points of maltodextrin, and can be added as needed according to the degree of branching of maltodextrin; αGP is used to hydrolyze the α-1,4 glycosidic bonds in maltodextrin to release glucose-1-phosphate; PGM is used to convert glucose-1-phosphate into glucose-6-phosphate; IPS is used to convert glucose-6-phosphate into inositol-1-phosphate.

[0136] The above IA, αGP, PGM and IPS are all thermostable enzymes derived from thermophilic microorganisms. These enzymes can withstand reaction temperatures of 55°C to 80°C. The thermostable temperature range of 55°C to 80°C means that the above enzymes can remain inactivated for a long time under this temperature condition and have high catalytic activity.

[0137] 1.1 Synthesis of recombinant plasmid

[0138] The sources and sequences of αGP, PGM, IPS, and IA are shown in Table 2:

[0139] Table 2 Enzyme sources and sequences

[0140] name source Amino acid sequence Nucleotide sequence α-Glucan phosphorylase Coprothermobacter sp. SEQ ID NO:1 SEQ ID NO:2 Phosphoglucomutase Thermostat maritima MSB8 SEQ ID NO:3 SEQ ID NO:4 Inositol-1-phosphate synthase Thermostat maritima MSB8 SEQ ID NO:5 SEQ ID NO:6 Isoamylase Sulfurisphaera tokodaii SEQ ID NO:7 SEQ ID NO:8

[0141] The nucleotide sequences shown in Table 2 were respectively inserted between the NdeI / HindII restriction sites of the pET28a plasmid to obtain different recombinant plasmid sequences, and four complete recombinant plasmids were synthesized by Sangon Biotechnology (Shanghai) Co., Ltd.

[0142] 1.2 Construction of recombinant Escherichia coli BL21 (DE3) strain containing genes encoding enzymes required for inositol-1-phosphate production

[0143] (1) Preparation of competent cells of E. coli BL21 (DE3): Streak a single colony of E. coli BL21 (DE3) stored at -80°C. Pick a single colony and inoculate it into 5 mL of LB medium. Cultivate it with shaking at 220 rpm at 37°C for about 3 h until the OD reaches 0. 600The cell culture temperature was about 0.5, and then the cultured bacterial solution was placed in an ice bath for 30 minutes; the bacterial solution obtained after the ice bath was transferred to a pre-cooled sterile centrifuge tube, centrifuged at 4000rpm for 10 minutes at 4°C, and the supernatant was discarded to collect the bacteria. The bacteria were resuspended in pre-cooled sterile water, centrifuged at 4000rpm for 10 minutes at 4°C, and the supernatant was discarded; the bacteria were resuspended twice in 0.1M CaCl2 solution, centrifuged at 4000rpm for 10 minutes at 4°C, and the supernatant was discarded; finally, the bacterial cells were resuspended in an appropriate amount of 0.1M CaCl2 solution containing 15% glycerol, and 100μL per tube was dispensed into 1.5mL centrifuge tubes, quickly placed in liquid nitrogen for quick freezing, and stored at -80°C.

[0144] (2) The four synthesized recombinant plasmids were added to 100 μL of E. coli BL21 (DE3) competent cells, with the added volume of each recombinant plasmid being 3 μL. The cells were placed on ice for 30 min, then heat-shocked at 42°C for 45 s, and immediately placed on ice for 2-5 min. After adding 800 μL of LB medium, the cells were placed on a 30°C shaker for incubation for 45 min, spread on a kanamycin-resistant LB plate (containing 50 μg / mL of kanamycin), and inverted in a 30°C incubator for overnight incubation. A single colony was picked and placed in a kanamycin-resistant liquid LB medium (containing 50 μg / mL of kanamycin). After several hours of incubation, glycerol with a final concentration of 30% was added and the recombinant strains were preserved at -80°C.

[0145] 1.3 Preparation of homogenized enzyme solution

[0146] (1) streaking the recombinant strains stored at -80°C in Example 1.2 to obtain single colonies;

[0147] (2) A single colony was picked and inoculated into 5 mL of kanamycin-resistant liquid LB medium (containing 50 μg / mL kanamycin) and cultured at 37°C with shaking at 220 rpm for 4 to 5 h.

[0148] (3) Transfer the bacterial solution in step (2) to a shake flask containing 150 mL of TB medium at a 1% (v / v) inoculation volume and culture at 37°C and 220 rpm until the OD 600 When the pH value was about 0.8, IPTG was added to a final concentration of 0.1 mM and cultured overnight at 25°C and 220 rpm to induce the expression of the enzyme protein.

[0149] (4) After the induction expression is completed, the wet cells are collected by centrifugation and resuspended in 0.1M potassium phosphate buffer (pH7.0) at a ratio of 1:6 (g / mL) to obtain a resuspension. The resuspension is then homogenized with a homogenizer at a homogenization pressure of 600 MPa and a homogenization time of 2 min to obtain a homogenized enzyme solution containing the desired enzyme protein.

[0150] (5) The molecular weights of the enzyme proteins in the four homogenized enzyme solutions were tested one by one using an SDS-PAGE kit. The test results are shown in Figure 1 The molecular weights of the four enzymes were 95.3 kDa (αGP), 46.8 kDa (PGM), 43.7 kDa (IPS), and 83.1 kDa (IA), which were consistent with the expected molecular weights, indicating that the four enzymes had been correctly expressed.

[0151] Example 2 Preparation of homogenized enzyme solution of phytase required for in vitro production of inositol

[0152] The inventor found in the study that phytase can remove the phosphate group from inositol-1-phosphate in vitro to obtain inositol, so phytase has certain catalytic activity for inositol-1-phosphate to generate inositol in vitro, but the catalytic activity of phytase from different sources is different, thereby affecting the final yield of inositol, therefore, the present embodiment has selected phytase from four different sources, and is formulated into a homogenized enzyme liquid for subsequent screening. The source and sequence of the 4 phytases of the present embodiment are as shown in Table 3:

[0153] Table 3 Sources and sequences of four phytases

[0154]

[0155] In Table 3, the suitable temperature range refers to the temperature range in which any of the above phytases catalyzes inositol-1-phosphate standard products to generate inositol in vitro alone, and has a higher inositol yield. The suitable pH range refers to the pH range in which any of the above phytases catalyzes inositol-1-phosphate standard products to generate inositol in vitro alone, and has a higher inositol yield.

[0156] The nucleotide sequences as shown in Table 3 are respectively inserted between the NdeI / HindII restriction sites of the pET28a plasmid to obtain different recombinant plasmid sequences, and 4 complete recombinant plasmids are synthesized by Shenggong Bioengineering (Shanghai) Co., Ltd. Then, a recombinant Escherichia coli BL21 (DE3) strain containing the coding gene of phytase is constructed according to Section 1.2 of Example 1, and a homogenized enzyme liquid containing phytase is prepared according to Section 1.3 of Example 1. The molecular weight of the enzyme protein in the 4 homogenized enzyme liquids containing phytase is detected one by one using an SDS-PAGE kit. The detection results about molecular weight are as follows: the molecular weight of phytase 1 is 55.4kDa, the molecular weight of phytase 2 is 49.4kDa, the molecular weight of phytase 3 is 47.9kDa, and the molecular weight of phytase 4 is 50.1kDa.

[0157] Example 3 Screening of phytases from different sources

[0158] In order to explore the effect of phytases from different sources on the yield of inositol in a multi-enzyme system, in this example, the homogenized enzyme solutions of phytases from different sources obtained in Example 2 and the homogenized enzyme solutions of heat-resistant αGP, PGM, IPS and IA obtained in Example 1 respectively constituted a multi-enzyme system, and the multi-enzyme system was used to prepare inositol using maltodextrin as a raw material. The details are as follows:

[0159] (1) The homogenized enzyme solutions of αGP, PGM, IPS, and IA obtained in Example 1 and the homogenized enzyme solutions of the four phytases obtained in Example 2 were centrifuged at 12,000 rpm for 5 min, and the precipitate (such as cell debris after homogenization) was discarded to obtain a primary supernatant; a portion of the primary supernatant was placed in a 60° C. metal bath and heated for 30 min to denature heat-sensitive impurities at high temperature, and then centrifuged again at 12,000 rpm for 5 min, and the precipitate (such as denatured impurities) was discarded to obtain a secondary supernatant, thereby obtaining 8 crude enzyme solutions respectively.

[0160] (2) Maltodextrin with a final concentration of 10 g / L, potassium phosphate buffer (pH 7.0) with a final concentration of 0.1 M, crude enzyme solutions of αGP, PGM, IPS, and IA, a crude phytase solution, and Mg with a final concentration of 8 mM were added to the reaction vessel in sequence. 2+ , forming a 1mL reaction system. A total of 4 reaction systems were obtained. Among them, the amount of crude enzyme solution added was: 200μL of αGP crude enzyme solution, 30μL of PGM crude enzyme solution, 400μL of IPS crude enzyme solution, 150μL of phytase crude enzyme solution (one of the four phytases), and 50μL of IA crude enzyme solution.

[0161] (3) The reaction system was placed in a 60° C. metal bath for 15 h, and samples were taken for HPLC testing. The test results are shown in Table 4.

[0162] Table 4 Catalytic results of four reaction systems

[0163] name Types of enzymes in the reaction system Inositol yield (g / L) Yield % Reaction system 1 αGP, PGM, IPS, IA, phytase 1 3.57 35.7 Reaction system 2 αGP, PGM, IPS, IA, phytase 2 2.92 29.2 Reaction system 3 αGP, PGM, IPS, IA, phytase 3 2.59 25.9 Reaction system 4 αGP, PGM, IPS, IA, phytase 4 1.28 12.8

[0164] In Table 4, the calculation formula of the yield is: yield = inositol production (g / L) / initial concentration of maltodextrin (g / L) × 100%.

[0165] The reaction system of this embodiment contains IA, and the role of IA is to hydrolyze the α-1,6 glycosidic bonds in maltodextrin, so that the remaining components basically contain α-1,4 glycosidic bonds, which can be further hydrolyzed by αGP to increase the yield of inositol. Other experimental results show that if the reaction system does not contain IA, inositol can still be produced, but because αGP cannot hydrolyze α-1,6 glycosidic bonds, the yield of inositol will be reduced relative to this embodiment. Depending on the number of α-1,6 glycosidic bonds contained in maltodextrin, the degree of reduction in inositol content is also different, generally there will be a reduction of 15% to 40%, so IA was added to the four reaction systems of this embodiment.

[0166] It can be seen from the experimental results in Table 4 that under the same reaction conditions, the reaction system containing phytase 1 has the highest inositol production and yield.

[0167] Example 4 Scale-up experiment of inositol production

[0168] This example involves an amplification experiment of a reaction system containing phytase 1. The volume of the reaction system involved in Example 3 was 1 mL, and this example amplified the reaction system to 60 mL.

[0169] Example 4-1

[0170] (1) The homogenized enzyme solutions of αGP, PGM, IPS and IA obtained in Example 1 and the homogenized enzyme solution of phytase 1 obtained in Example 2 were centrifuged at 12000 rpm for 5 min, and the precipitate (such as cell debris after homogenization) was discarded to obtain a primary supernatant; a portion of the primary supernatant was placed in a 60° C. metal bath and heated for 30 min to denature heat-sensitive impurities at high temperature, and centrifuged again at 12000 rpm for 5 min, and the precipitate (such as denatured impurities) was discarded to obtain a secondary supernatant, thereby obtaining crude enzyme solutions.

[0171] (2) Add maltodextrin with a final concentration of 150 g / L, potassium phosphate buffer with a final concentration of 0.1 M (pH 7.0), crude enzyme solution, and MgCl with a final concentration of 8 mM into the reaction vessel in sequence. 2+ , forming a 60mL reaction system. The amount of crude enzyme solution added is: 3mL of αGP crude enzyme solution, 1.2mL of PGM crude enzyme solution, 18mL of IPS crude enzyme solution, 22.5mL of phytase 1 crude enzyme solution and 2.5mL of IA crude enzyme solution.

[0172] (3) The reaction system was placed in an oil bath set at 65°C (the actual temperature of the reaction system was between 60°C and 63°C), and magnetic stirring (300 rpm) was used to mix the reaction system more evenly. Samples were taken after 15h and 30h of reaction and HPLC detection was performed. The results showed that after 15h of reaction, the inositol production was 75.33 g / L and the yield was 50.22%; after 30h of reaction, the inositol production reached 146.76 g / L and the yield reached 97.84%.

[0173] Example 4-2

[0174] Different from Example 4-1, the final concentration of maltodextrin added in Example 4-2 was 180 g / L, and the other conditions were the same as those in Example 4-1. After 15 hours of reaction, the yield of inositol was 89.47 g / L, with a yield of 49.71%, and after 30 hours of reaction, the yield of inositol was 142.34 g / L, with a yield of 79.08%.

[0175] Example 4-3

[0176] Different from Example 4-1, the final concentration of maltodextrin added in Example 4-3 was 100 g / L, and the other conditions were the same as those in Example 4-1. After 15 hours of reaction, the yield of inositol was 49.57 g / L, and the yield was 49.57%. After 30 hours of reaction, the yield of inositol was 96.53 g / L, and the yield was 96.53%.

[0177] The reaction parameters and reaction results of the above embodiments are shown in Table 5.

[0178] Table 5 Reaction parameters and reaction results of Example 4

[0179]

[0180] In Table 5, the initial concentration of maltodextrin in the reaction system refers to the concentration of maltodextrin when it is first added to the reaction system, that is, the concentration when the reaction time is 0 (also known as the working concentration of maltodextrin), rather than the mother liquor concentration of maltodextrin. According to the results of Table 5, for the reaction system containing phytase 1, when the initial concentration of maltodextrin is 150g / L, the output and yield of inositol are very high when the reaction is 30h, which has industrial application prospects. When the initial concentration of maltodextrin is higher or lower, the final output and yield are relatively low.

[0181] According to the above experimental results, the reaction system containing phytase 1 has high production and yield of inositol in both small-scale and scale-up experiments, and is suitable for industrial production.

[0182] The amino acid sequence and nucleotide sequence corresponding to the enzyme used in the present invention are:

[0183] SEQ ID NO:1

[0184] 。

[0185] SEQ ID NO:2

[0186]

[0187] SEQ ID NO:3

[0188] MKYFGTDGIRGIFGETLTDELAFKVGKALGEIVGEGKVIVGKDTRVSGDSLEAAISAGLTSMGVDVLLCGILPTPAVALLTRITRSFGVVISASHNPPEYNGIKVLKGGYKIPDEMEAEIEKRLENGSFLTRSVVGRTKSFREGRDMYIGAVLEMFRDLDLTGEMVSLDLANGATTTTAREVFEFLGAKVEVFNDSQDGLLINQGCGATHPRFLAEEMKNGKVGFTFDGDGDRVIAVDEERNVVNGDRIIGILAVGLKEEGRLNSDTVVGTVMTNGGLEDFLKEKGIRLLRTKVGDKYVLEKMLESGANLGGERSGHIIILDRSTTGDGLITALELMRVLKRSGRKLSDFAKEIPDYPQITKNVRRTERMSLENENLRKIVEESTSRGYRVVIRPSGTEPVIRITVEGKDREEIEKIVEEISRVLES。

[0189] SEQ ID NO:4

[0190]

[0191] SEQ ID NO:5

[0192] MDRLDFSIKLLRKVGHLLMIHWGRVDNVEKKTGFKDIVTEIDREAQRMIVDEIRKFFPDENIMAEEGIFEKGDRLWIIDPIDGTINFVHGLPNFSISLAYVENGEVKLGVVHAPALNETLYAEEGSGAFFNGERIRVSENASLEECVGSTGSYVDFTGKFIERMEKRTRRIRILGSAAALNAAYVGAGRVDFFVTWRINPWDIAAGLIIVKEAGGMVTDFSGKEANAFSKNFIFSNGLIHDEVVKVVNEVVEEIGGK.

[0193] SEQ ID NO:6

[0194] ATGGATCGCCTGGACTTCAGCATCAAACTGCTGCGTAAAGTTGGCCACCTGCTGATGATCCACTGGGGTCGTGTTGATAACGTTGAAAAGAAAACCGGTTTCAAAGATATCGTTACCGAAATCGATCGTGAAGCGCAGCGTATGATCGTTGATGAAATCCGTAAATTCTTCCCGGATGAAAACATCATGGCGGAAGAAGGTATCTTCGAAAAAGGTGACCGTCTGTGGATCATCGACCCGATCGATGGCACCATCAACTTCGTTCACGGCCTGCCGAACTTCAGCATCTCTCTGGCGTACGTTGAAAACGGCGAAGTTAAACTGGGCGTTGTTCACGCGCCGGCGCTGAACGAAACCCTGTACGCGGAAGAAGGTAGCGGCGCGTTCTTCAACGGTGAACGTATCCGTGTTAGCGAAAACGCGTCCCTGGAAGAATGCGTTGGCTCTACCGGCAGCTACGTTGATTTCACCGGTAAATTCATCGAACGTATGGAAAAACGTACCCGTCGTATCCGTATCCTGGGCAGCGCAGCGCTGAACGCAGCGTACGTTGGTGCGGGTCGTGTTGATTTCTTCGTTACCTGGCGCATCAACCCGTGGGATATCGCTGCGGGCCTGATCATCGTGAAAGAAGCGGGTGGCATGGTTACCGATTTCAGCGGTAAAGAAGCGAACGCGTTCAGCAAAAACTTCATCTTCAGCAACGGCCTGATCCATGATGAAGTTGTTAAAGTGGTGAACGAAGTTGTTGAAGAAATCGGCGGCAAATAA。

[0195] SEQ ID NO:7

[0196] MVFSHKDRPLRPGEPYPLGANWEEEDDGVNFSIFSENATKVELLIYSPTNQKYPKEVIEVKQRSGDIWHVFVPGLGPGTLYAYRIYGPYKPDQGLRFNPNKVLIDPYAKAINGTLNWNDAVFGYKIGDSNQDLSFDDRPDDEFIPKGVVINPYFEWDDDHFFRRKKIPLKDTIIYEVHVKGFTKLRPDLPENIRGTYKGFASRQMIEYLKDLGVTTVEIMPVQQFVDDRFLVEKGLRNYWGYNPINYFSPECRYSSSGCMGEQVNEFKEMVNELHNAGFEVIIDVVYNHTAEGNHLGPTLSFRGIDNLAYYMLVPDNKRYYLDFTGTGNTLNLSHPRVLQMVLDSLRYWVLEMHVDGFRFDLAAALARQLYSVNMLSTFFVAIQQDPVLSQVKLIAEPWDVGPGGYQVGNFPYLWAEWNGKYRDTIRRFWRGEAIPYEELANRLMGSPDLYAGNNKTPFASINYITSHDGFTLEDLVSYNQKHNEANGFNNQDGMNENYSWNCGVEGETNDANVIQCREKQKRNFIITLFVSQGVPMILGGDELSRTQRGNNNAFCQDNEISWFNWNLDERKQRFHDFVRSMIYFYRAHPIFRRERYFQGKKLHGMPLKDVTFLKPDGNEADEQTWKSPTNFIAYILEGSVIDEVNDRGERIADDSFLIILNGSPNNIKFKFPQGKWSLVVSSYLRELRDDERVVDGGKELEIEGRTAMVYRRIEY。

[0197] SEQ ID NO:8

[0198]

[0199] SEQ ID NO:9

[0200] MKKLYNGRRVRRGEDGFPIRFNDGGIATLIVGIMVTLTFLLSAAYLLSGRVSAAPSSAGSKSCDTVDLGYQCSPATSHLWGQYSPFFSLEDELSVSSKLPKDCRITLVQVLSRHGARYPTSSKSKKYKKLVTAIQANATDFKGKFAFLKTYNYTLGADDLTPFGEQQLVNSGIKFYQRYKALARSVVPFIRASGSDRVIASGEKFIEGFQQAKLADPGATNRAAPAISVIIPESETFNNTLDHGVCTKFEASQLGDEVAANFTALFAPDIRARAEKHLPGVTLTDEDVVSLMDMCSFDTVARTSDASQLSPFCQLFTHNEWKKYNYLQSLGKYYGYGAGNPLGPAQGIGFTNELIARLTRSPVQDHTSTNSTLVSNPATFPLNATMYVDFSHDNSMVSIFFALGLYNGTEPLSRTSVESAKELDGYSASWVVPFGARAYFETMQCKSEKEPLVRALINDRVVPLHGCDVDKLGRCKLNDFVKGLSWARSGGNWGECFS。

[0201] SEQ ID NO:10

[0202]

[0203] SEQ ID NO:11

[0204] MTIAKEYLRLSILTLVLSSFTLSAAPLAAQSTGYTLERVVILSRHGVRSPTKQTQLMNDVTPDKWPQWPVKAGYLTPRGAGLVTLMGGFYGDYFRSYGLLPAGCPADESIYVQADVDQRTRLTGQAFLDGIAPDCGLKVHYQADLKKIDPLFHTVEAGVCKLDPEKTHQAVEKRLGGPLNELSQRYAKPFALMGEVLNFSASPYCNSLQQKGKTCDFATFAANEIEVNKEGTKVSLSGPLALSSTLGEIFLLQNSQAMPDVAWNRLSGEENWISLLSLHNAQFDLMAKTPYIARHKGTPLLQQIDTALVLQRDAQGQTLPLSPQTKLLFLGGHDTNIANIAGMLGANWQLPQQPDNTPPGGGLVFELWQNPDNHQRYVAVKMFYQTMEQLRNADKLDLKNNPARIVPIAIEGCENEGDNKLCQLETFQKKVAQVIEPACHI。

[0205] SEQ ID NO:12

[0206]

[0207] SEQ ID NO:14

[0208]

[0209] SEQ ID NO:15

[0210] LAVPASRNQSTCDTVDQGYQCFSETSHLWGQYAPFFSLANKSAISPDVPAGCQVTFAQVLSRHGARYPTDSKGKKYSALIEEIQQNATTFEEKYAFLKTYNYSLGADDLTPFGEQELVYSGVKFYQRYESLTRNIVPFIRSSGSSRVIASGNKFIEGFQSTKLKDPRAQPGQSSPKIDVVISEASTSNNTLDPGTCTVFEDSELADDIEANFTATFVPSIRQRLENDLSGVTLTDTEVTYLMDMCSFDTISTSTVDTKLSPFCDLFTHEEWINYDYLQSLNKYYGHGAGNPLGPTQGVGYANELIARLTHSPVHDDTSSNHTLDSNPATFPLNSTLYADFSHDNGIISILFALGLYNGTKPLSSTTAENITQTDGFSSAWTVPFASRMYVEMMQCQSEQEPLVRVLVNDRVVPLHGCPVDALGRCTRDSFVKGLSFARSGGDWAECFA。

[0211] SEQ ID NO:16

[0212]

Claims

1. A method for preparing inositol, characterized in that: The conversion of maltodextrin into inositol was performed using a multienzyme system; The multi-enzyme system comprises alpha-glucan phosphorylase, phosphoglucomutase, inositol-1-phosphate synthase and phytase.

2. The preparation method according to claim 1, characterized in that The amino acid sequence of the α-glucan phosphorylase is shown in SEQ ID NO: 1; and / or, The amino acid sequence of the phosphoglucomutase is shown in SEQ ID NO: 3; and / or, The amino acid sequence of the inositol-1-phosphate synthase is shown in SEQ ID NO: 5; and / or, The amino acid sequence of the phytase is selected from any one or more of SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13 and SEQ ID NO:15; Preferably, the nucleotide sequence encoding the α-glucan phosphorylase is shown in SEQ ID NO: 2; and / or, The nucleotide sequence encoding the phosphoglucomutase is shown in SEQ ID NO:4; and / or, The nucleotide sequence encoding the inositol-1-phosphate synthase is shown in SEQ ID NO: 6; and / or, The nucleotide sequence encoding the phytase is selected from any one or more of SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14 and SEQ ID NO:

16.

3. The preparation method according to claim 1, characterized in that: The multi-enzyme system also includes isoamylase; Preferably, the amino acid sequence of the isoamylase is as shown in SEQ ID NO: 7; More preferably, the nucleotide sequence encoding the isoamylase is shown as SEQ ID NO:

8.

4. The preparation method according to any one of claims 1 to 3, characterized in that: In the reaction system consisting of at least the multi-enzyme system and the maltodextrin, the initial concentration of the maltodextrin is 10 to 190 g / L, preferably 100 to 180 g / L; and / or, The conversion is carried out at a temperature of 58°C to 65°C, preferably 60°C to 63°C; and / or, The conversion is carried out in a phosphate buffer having a pH value of 6.9 to 7.1; preferably, the phosphate buffer is a potassium phosphate buffer; more preferably, the pH value of the potassium phosphate buffer is 7.0; and / or, Mg is added before or during the conversion. 2+ Preferably, the Mg 2+ The initial concentration is 7 mM to 9 mM, for example 8 mM; and / or, The conversion is carried out under shaking or stirring; preferably, the stirring is magnetic stirring, and / or the stirring speed is 300 rpm to 600 rpm; and / or, The duration of the conversion is 15 h to 36 h, preferably 28 h to 30 h.

5. A method for preparing a multi-enzyme system, characterized in that: It includes the following steps: After mixing α-glucan phosphorylase, phosphoglucomutase, inositol-1-phosphate synthase and phytase, a multi-enzyme system is obtained; Preferably, the amino acid sequence of the α-glucan phosphorylase is as shown in SEQ ID NO: 1; and / or, The amino acid sequence of the phosphoglucomutase is shown in SEQ ID NO: 3; and / or, The amino acid sequence of the inositol-1-phosphate synthase is shown in SEQ ID NO: 5; and / or, The amino acid sequence of the phytase is selected from any one or more of SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13 and SEQ ID NO:15; More preferably, the nucleotide sequence encoding the α-glucan phosphorylase is as shown in SEQ ID NO: 2; and / or, The nucleotide sequence encoding the phosphoglucomutase is shown in SEQ ID NO:4; and / or, The nucleotide sequence encoding the inositol-1-phosphate synthase is shown in SEQ ID NO: 6; and / or, The nucleotide sequence encoding the phytase is selected from any one or more of SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14 and SEQ ID NO:

16.

6. The preparation method according to claim 5, characterized in that: The α-glucan phosphorylase, the phosphoglucomutase, the inositol-1-phosphate synthase and the phytase are present alone or together in the form of whole-cell enzyme, homogenized enzyme solution, crude enzyme solution or solid enzyme powder; Preferably, any one or more of the α-glucan phosphorylase, the phosphoglucomutase, the inositol-1-phosphate synthase and the phytase are present in the form of a homogenized enzyme solution, and the ratio of the added mass of the wet cells to the added volume of the resuspension in the homogenized enzyme solution is 1 g: (5-10) mL; More preferably, the homogenized enzyme solution is heated before the mixing, and the heating temperature is 58°C to 65°C; preferably, 60°C to 63°C; and / or, the heating time is 30min to 60min, preferably 30min to 35min; Even more preferably, the resuspension liquid is water, buffer or culture medium.

7. The preparation method according to claim 5, characterized in that: During or after mixing, heat treated; Preferably, the heat treatment temperature is 58°C to 65°C; preferably, 60°C to 63°C; and / or, The heat treatment time is 30 min to 60 min, preferably 30 min to 35 min.

8. The preparation method according to claim 7, characterized in that: In the multi-enzyme system, the addition amounts of the α-glucan phosphorylase, the phosphoglucomutase, the inositol-1-phosphate synthase and the phytase are calculated according to the addition mass of the wet cells as follows: The added mass ratio of the wet bacteria capable of producing the α-glucan phosphorylase to the wet bacteria capable of producing the phosphoglucomutase is 1:(0.15-0.4); and / or, The added mass ratio of the wet bacteria capable of producing the α-glucan phosphorylase to the wet bacteria capable of producing the inositol-1-phosphate synthase is 1:(2-6); and / or, The mass ratio of the wet bacteria that can produce the α-glucan phosphorylase to the wet bacteria that can produce the phytase is 1:(0.75-7.5).

9. The preparation method according to any one of claims 5 to 8, characterized in that: The method further comprises: before, during or after the mixing, adding isoamylase; Preferably, the amino acid sequence of the isoamylase is as shown in SEQ ID NO: 7; and / or, the isoamylase is in the form of whole cell enzyme, homogenized enzyme solution, crude enzyme solution or solid enzyme powder; More preferably, the nucleotide sequence encoding the isoamylase is as shown in SEQ ID NO: 8; and / or, under the condition that the isoamylase is present in the form of the homogenized enzyme solution, it is heated before the mixing, and / or, it is heat-treated during or after the mixing; and / or In the multi-enzyme system, the addition amount of the α-glucan phosphorylase and the isoamylase is calculated according to the added mass of the wet cells as follows: the mass ratio of the wet cells capable of producing the α-glucan phosphorylase to the wet cells capable of producing the isoamylase is 1:(0.25-0.85); Further preferably, the temperature of the heating and / or heat treatment is 58°C to 65°C; preferably, 60°C to 63°C; and / or, The heating and / or heat treatment time is 30 min to 60 min, preferably 30 min to 35 min.

10. A multi-enzyme system, characterized in that: The multi-enzyme system comprises α-glucan phosphorylase, phosphoglucomutase, inositol-1-phosphate synthase and phytase; Preferably, the α-glucan phosphorylase, the phosphoglucomutase, the inositol-1-phosphate synthase and the phytase are present alone or together in the form of whole-cell enzyme, homogenized enzyme solution, crude enzyme solution or solid enzyme powder; and / or The multi-enzyme system further comprises isoamylase; and / or The amino acid sequence of the α-glucan phosphorylase is shown in SEQ ID NO: 1; and / or, The amino acid sequence of the phosphoglucomutase is shown in SEQ ID NO: 3; and / or, The amino acid sequence of the inositol-1-phosphate synthase is shown in SEQ ID NO: 5; and / or, The amino acid sequence of the phytase is selected from any one or more of SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13 and SEQ ID NO:15; More preferably, the isoamylase is in the form of whole-cell enzyme, homogenized enzyme solution, crude enzyme solution or solid enzyme powder; and / or, The amino acid sequence of the isoamylase is shown in SEQ ID NO: 7; and / or, The nucleotide sequence encoding the α-glucan phosphorylase is shown in SEQ ID NO: 2; and / or, The nucleotide sequence encoding the phosphoglucomutase is shown in SEQ ID NO:4; and / or, The nucleotide sequence encoding the inositol-1-phosphate synthase is shown in SEQ ID NO: 6; and / or, The nucleotide sequence encoding the phytase is selected from any one or more of SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14 and SEQ ID NO: 16; Even more preferably, the nucleotide sequence encoding the isoamylase is shown as SEQ ID NO:

8.

11. Use of the multi-enzyme system according to claim 10, or the multi-enzyme system obtained by the preparation method according to any one of claims 5 to 9, in catalyzing maltodextrin to produce inositol.

12. Use of a phytase in catalyzing the production of inositol from inositol-1-phosphate, wherein the amino acid sequence of the phytase is selected from any one or more of SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13 and SEQ ID NO: 15; Preferably, the inositol-1-phosphate is obtained by catalyzing α-glucan phosphorylase, phosphoglucomutase and inositol-1-phosphate synthase in sequence or together with maltodextrin as substrate; or, the inositol-1-phosphate is obtained by catalyzing maltodextrin by isoamylase, α-glucan phosphorylase, phosphoglucomutase and inositol-1-phosphate synthase in sequence or together with maltodextrin as substrate; More preferably, the amino acid sequence of the α-glucan phosphorylase is as shown in SEQ ID NO: 1; and / or, The amino acid sequence of the phosphoglucomutase is shown in SEQ ID NO: 3; and / or, The amino acid sequence of the inositol-1-phosphate synthase is shown in SEQ ID NO: 5; and / or, The amino acid sequence of the isoamylase is shown in SEQ ID NO: 7; Further more preferably, the nucleotide sequence encoding the α-glucan phosphorylase is as shown in SEQ ID NO: 2; and / or, The nucleotide sequence encoding the phosphoglucomutase is shown in SEQ ID NO:4; and / or, The nucleotide sequence encoding the inositol-1-phosphate synthase is shown in SEQ ID NO: 6; and / or, The nucleotide sequence encoding the isoamylase is shown in SEQ ID NO: 8; and / or, The nucleotide sequence encoding the phytase is selected from any one or more of SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14 and SEQ ID NO:

16.

13. An enzyme catalyzed reaction system, characterized in that: The enzyme catalytic reaction system comprises: maltodextrin, α-glucan phosphorylase, glucose phosphomutase, inositol-1-phosphate synthase and phytase; and a buffer and Mg 2+ One or two of; preferably the buffer is a phosphate buffer such as a potassium phosphate buffer; more preferably the pH of the buffer is 6.9 to 7.1, such as 7.0; Preferably, the enzyme catalyzed reaction system further comprises inositol and / or isoamylase; and / or, The amino acid sequence of the α-glucan phosphorylase is shown in SEQ ID NO: 1; and / or, The amino acid sequence of the phosphoglucomutase is shown in SEQ ID NO: 3; and / or, The amino acid sequence of the inositol-1-phosphate synthase is shown in SEQ ID NO: 5; and / or, The amino acid sequence of the phytase is selected from any one or more of SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13 and SEQ ID NO: 15; and / or, The initial concentration of maltodextrin in the enzyme-catalyzed reaction system is 10 to 190 g / L; More preferably, the nucleotide sequence encoding the α-glucan phosphorylase is as shown in SEQ ID NO: 2; and / or, The nucleotide sequence encoding the phosphoglucomutase is shown in SEQ ID NO:4; and / or, The nucleotide sequence encoding the inositol-1-phosphate synthase is shown in SEQ ID NO: 6; and / or, The nucleotide sequence encoding the phytase is selected from any one or more of SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14 and SEQ ID NO: 16; and / or, The amino acid sequence of the isoamylase is shown in SEQ ID NO: 7; and / or, The initial concentration of maltodextrin in the enzyme-catalyzed reaction system is 100-180 g / L; Further more preferably, the nucleotide sequence encoding the isoamylase is shown as SEQ ID NO:

8.

14. A reaction end product system, characterized in that: The reaction end product system comprises: Maltodextrin; α-glucan phosphorylase, which can hydrolyze the α-1,4 glycosidic bonds in the maltodextrin to release glucose-1-phosphate; Phosphoglucomutase, capable of converting the glucose-1-phosphate into glucose-6-phosphate; Inositol-1-phosphate synthase, capable of converting the glucose-6-phosphate into inositol-1-phosphate; a phytase capable of converting the inositol-1-phosphate into inositol; and Inositol, wherein the mass volume concentration (g / L) of the inositol in the final reaction product system accounts for more than 79% of the initial mass volume concentration (g / L) of maltodextrin; Preferably, the mass volume concentration (g / L) of the inositol in the final reaction product system accounts for more than 96% of the initial mass volume concentration (g / L) of the maltodextrin; and / or, The amino acid sequence of the α-glucan phosphorylase is shown in SEQ ID NO: 1; and / or, The amino acid sequence of the phosphoglucomutase is shown in SEQ ID NO: 3; and / or, The amino acid sequence of the inositol-1-phosphate synthase is shown in SEQ ID NO: 5; and / or, The amino acid sequence of the phytase is selected from any one or more of SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13 and SEQ ID NO: 15; and / or, The reaction end product system also includes: isoamylase, which can hydrolyze the α-1,6 glycosidic bonds of the branching points of the maltodextrin; More preferably, the mass volume concentration (g / L) of the inositol in the final reaction product system accounts for more than 97% of the initial mass volume concentration (g / L) of the maltodextrin; and / or, The nucleotide sequence encoding the α-glucan phosphorylase is shown in SEQ ID NO: 2; and / or, The nucleotide sequence encoding the phosphoglucomutase is shown in SEQ ID NO:4; and / or, The nucleotide sequence encoding the inositol-1-phosphate synthase is shown in SEQ ID NO: 6; and / or, The nucleotide sequence encoding the phytase is selected from any one or more of SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14 and SEQ ID NO: 16; and / or, The amino acid sequence of the isoamylase is shown in SEQ ID NO: 7; Further more preferably, the nucleotide sequence encoding the isoamylase is shown as SEQ ID NO:8.

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