Preparation method of inositol

Abstract

The invention provides an application of inositol monophosphate in an enzyme catalysis reaction system for preparing inositol. The invention also provides a preparation method of the inositol, the starch is used as a substrate, glucan phosphorylase, glucose phosphate mutase, inositol-3-phosphate synthase and inositol monophosphate are added as enzyme catalysts to catalyze the substrate to generate the inositol, the inositol monophosphate is derived from Archaeoglobus fulgidus, the amino acid sequence is shown as SEQ ID NO: 12, or the amino acid sequence is shown as SEQ ID NO: 1, or the amino acid sequence is shown as SEQ ID NO: 2, or the amino acid sequence is shown as SEQ ID NO: 3, or the amino acid sequence is shown as SEQ ID NO: 4, or the amino acid sequence is shown as SEQ ID NO: 5, or the amino acid sequence is shown as SEQ ID NO: 6. The inositol monophosphate is derived from Thermococcus kodakarensis, and the amino acid sequence of the inositol monophosphate is as shown in SEQ ID NO: 13; or the inositol monophosphate is derived from Pseudothermotoga lettinae, and the amino acid sequence of the inositol monophosphate is as shown in SEQ ID NO: 14. Inositol-3-phosphate synthetase and inositol monophosphate with good performance are screened, high-concentration starch is efficiently catalyzed to be converted into inositol by a one-pot method in a whole-cell feeding mode, and NAD < + > and other coenzymes do not need to be used. Under a 60L reaction system, 200g / L of corn starch is catalyzed, the unit yield of the prepared inositol reaches 161g / L, and the method has a good industrial application prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to a method for preparing myo-inositol, and belongs to the field of enzyme catalysis. BACKGROUND

[0002] Myo-inositol (MI), also known as cyclohexanepentol, is a direct precursor of glucuronic acid and is an essential substance for the growth of humans, animals and microorganisms. Myo-inositol is widely used as a nutritional supplement in the pharmaceutical, food, feed and other industries. In recent years, with the continuous development of the downstream market, the demand for myo-inositol has shown an increasing trend year by year.

[0003] At present, the production processes of myo-inositol mainly include pressurized hydrolysis, atmospheric hydrolysis, enzymatic hydrolysis, microbial fermentation and in vitro enzyme synthesis methods. Among them, the microbial fermentation method for producing myo-inositol is based on the theoretical research on the myo-inositol metabolic pathway of yeast. Shirai Makoto team of Japan and Pramod Agmwal of the United States and others have made important explorations. Shirai Makoto team has 8 patents related to the production of myo-inositol by microbial fermentation method in the 1990s, which were collected by the European Patent Office.

[0004] The in vitro enzyme synthesis method is a method for preparing and synthesizing myo-inositol by step or one-step through in vitro multi-enzyme catalytic pathway based on the research of myo-inositol metabolic pathway. The in vitro enzyme synthesis method has the advantages of environmental protection and high myo-inositol yield in production process. However, the existing production processes have problems such as unstable enzyme activity, high cost, low raw material utilization rate and low yield to varying degrees, which have great limitations.

[0005] PCT application with publication number WO2014197702A1 discloses the reaction of using α-glucan phosphorylase and phosphoglucomutase to produce glucose-6-phosphate (G6P) from starch or maltodextrin. Todd B. Reynolds et al., Microbiology (2009), 155, 1386-1396, discloses a production method for converting glucose-6-phosphate (G6P) to myo-inositol-3-phosphate by myo-inositol-3-phosphate synthase and then converting myo-inositol-3-phosphate to myo-inositol by myo-inositol monophosphatase in vitro.

[0006] Patent document CN106148425A discloses a method for producing myo-inositol by in vitro multi-enzyme one-step catalysis of starch and starch derivatives, but the starch used in the method embodiment is soluble starch, and the substrate concentration is very low, and the separation and purification steps of the multi-enzyme system related enzymes are complicated. Chun You et al. (An In Vitro Synthetic Biology Platform for the Industrial Biomanufacturing of Myo-Inositol From Starch. Biotechnol. Bioeng. 2017, 114: 1855-1864.) reported a cell-free, coenzyme-free (such as ATP or NAD⁺) four-enzyme catalytic pathway, which realized one-step conversion of starch to myo-inositol. In this method, all enzymes are derived from hyperthermophilic microorganisms, which can be expressed by high-density fermentation of Escherichia coli, but need to be simply purified by heat precipitation, and after scaling up the reaction scale, the highest substrate concentration is only 125 g / L, and the final myo-inositol yield per unit volume is not high.

[0007] Patent document CN118028276A also discloses a myo-inositol-3-phosphate synthase mutant, a preparation method, application and preparation method of myo-inositol. The method can perform enzymatic reaction on maltodextrin in the presence of myo-inositol-3-phosphate synthase mutant and myo-inositol monophosphatase, and optionally maltodextrin phosphorylase and glucose phosphoric transposase. But this method is only at the laboratory small test level, and also has the problem of low raw material utilization rate.

[0008] In summary, the existing in vitro enzyme catalytic conversion method for preparing myo-inositol has less selectivity of enzymes, and is mostly at the laboratory small test level, which has great limitations in industrial production application. Therefore, it is urgent to develop a new method which can use whole cells, has high performance, high yield, simple production process, low cost, is suitable for high substrate concentration and high product myo-inositol output, and is easy to scale up myo-inositol preparation. SUMMARY

[0009] The existing in vitro multi-enzyme catalytic reaction method for preparing myo-inositol has limitations in industrial production, such as complicated enzyme purification steps, complex production process, and low substrate concentration. The enzyme selectivity used in enzyme conversion method is small, which is not suitable for the preparation and application of myo-inositol.

[0010] The application provides application of myo-inositol monophosphatase in an enzyme catalysis reaction system for preparing myo-inositol, wherein the myo-inositol monophosphatase is derived from Archaeoglobus fulgidus, and the amino acid sequence is shown as SEQ ID NO: 12; or the myo-inositol monophosphatase is derived from Thermococcus kodakarensis, and the amino acid sequence is shown as SEQ ID NO: 13; or the myo-inositol monophosphatase is derived from Pseudothermotoga lettingae, and the amino acid sequence is shown as SEQ ID NO: 14.

[0011] The application provides a preparation method of myo-inositol, which is prepared by taking starch as a substrate, adding glucan phosphorylase, phosphoglucomutase, myo-inositol-3-phosphate synthase and myo-inositol monophosphatase as enzyme catalysts to catalyze the substrate to generate myo-inositol; the myo-inositol monophosphatase is derived from Archaeoglobus fulgidus, and the amino acid sequence is shown as SEQ ID NO: 12; or the myo-inositol monophosphatase is derived from Thermococcus kodakarensis, and the amino acid sequence is shown as SEQ ID NO: 13; or the myo-inositol monophosphatase is derived from Pseudothermotoga lettingae, and the amino acid sequence is shown as SEQ ID NO: 14.

[0012] The myo-inositol-3-phosphate synthase is derived from Thermococcus kodakarensis, and the amino acid sequence is shown as SEQ ID NO: 6.

[0013] The myo-inositol-3-phosphate synthase is derived from Pyrococcus yayanosii, and the amino acid sequence is shown as SEQ ID NO: 7.

[0014] The myo-inositol-3-phosphate synthase is derived from Pyrococcus abyssi, and the amino acid sequence is shown as SEQ ID NO: 8.

[0015] The enzyme catalysts further comprise isoamylase.

[0016] The preparation method comprises the following steps: a. starch is weighed, and glucan phosphorylase, phosphoglucomutase, myo-inositol-3-phosphate synthase and myo-inositol monophosphatase are added; b. catalysis reaction is carried out at 60-90 DEG C for 10-120 hours, and myo-inositol is obtained.

[0017] The starch used in the reaction system in step a is 10-250 g / L; The enzyme catalytic reaction temperature in step b is 75-80°C, preferably, the enzyme catalytic reaction temperature is 80°C; and the reaction time is 20-80 hours.

[0018] The myo-inositol-3-phosphate synthase and the myo-inositol monophosphatase are in the form of primary purified enzyme solution, homogeneous enzyme solution or whole cell enzyme.

[0019] The inventors screened wild-type myo-inositol-3-phosphate synthase and myo-inositol monophosphatase with good performance, which can be synergistically catalyzed with glucan phosphorylase and phosphoglucomutase to efficiently catalyze high-concentration starch to myo-inositol in one-pot method in the form of whole cell feed, without using NAD + coenzymes. In a 60 L reaction system, 200 g / L of corn starch is catalyzed to prepare myo-inositol with a unit yield of 161 g / L, and the reaction yield reaches 80.5%, which has good industrial application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 The catalytic conversion path for preparing myo-inositol from starch as a substrate by a multi-enzyme system; Figure 2 LC-MS detection results of the multi-enzyme system composed of isoamylase A, glucan phosphorylase B, phosphoglucomutase C, myo-inositol monophosphatase E1 and myo-inositol-3-phosphate synthase D4 in the catalytic reaction for preparing myo-inositol under a corn starch concentration of 150 g / L. DETAILED DESCRIPTION

[0021] The embodiments of the present application are described in detail below, and the examples of the embodiments are intended to explain the present application, and cannot be understood as a limitation of the present application.

[0022] The specific techniques or conditions not specified in the examples are carried out according to the techniques or conditions described in the literature in the art or according to the product instructions. The various materials and reagents used in the examples are materials and reagents commonly used in the art, which can be obtained through conventional commercial channels, and the main materials and reagents are shown in Table 1: Table 1 List of main materials and reagents Name Supplier Product No. Escherichia coli BL21 (DE3) Beijing Qianke Biotechnology Co., Ltd. DZC201-96B100 pET28a plasmid Wuhan Moliang Biotechnology Co., Ltd. P0023 Myo-inositol standard Shanghai Macklin Biochemical Technology Co., Ltd. I811835 Corn starch Shanghai Macklin Biochemical Technology Co., Ltd. S818265 Magnesium chloride hexahydrate Shanghai Macklin Biochemical Technology Co., Ltd. M813913 During the implementation of the present application, the culture of E. coli can be carried out in a shake flask using conventional LB or TB medium, or can be carried out in a large-scale fermentation device using a cheaper corn slurry medium. The formulations of TB medium and LB medium are as follows: TB medium formula: Tryptone 12 g / L; yeast extract 24 g / L; glycerol 4 mL / L; potassium phosphate dibasic 16.43 g / L; potassium phosphate monobasic 2.31 g / L.

[0023] LB medium formula: Tryptone 10 g / L, yeast powder 5 g / L, NaCl 10 g / L.

[0024] The LC-MS detection method is as follows: The inositol is detected by LC-MS, the chromatographic column is a phenomenex Gemini® 5 μm C18 column, the mobile phase is acetonitrile: water = 95:5, the flow rate is 1 mL / min, the column temperature is 35°C, and the injection volume is 1 μL. The inositol standard sample is dissolved in water to prepare standard sample solutions with different concentrations. The retention time of the inositol control sample is about 2.689 min. The characteristic ion m / z = +181.1 of inositol is extracted under the SIM mode (Selected Ion Monitoring mode), the Mass response peak area of the ion is taken, a concentration-peak area curve is drawn, and the inositol concentration in the detection sample is calculated according to the standard curve.

[0025] The calculation method of the yield is as follows: After the catalytic reaction is completed, the inositol concentration in the reaction solution is calculated according to the above method, so as to calculate the reaction yield = (inositol concentration x reaction system volume) / starch feed amount x 100%.

[0026] The biosynthesis pathway of inositol prepared by in vitro multi-enzyme catalytic conversion of starch as a substrate is as shown in Figure 1 The last two steps of enzyme catalytic reaction are irreversible, which has a crucial influence on the reaction. In addition, the annotation of isozymes in the database cannot be equivalent to the same biological activity. Even if the functional annotation is the same, the enzymes from different sources have differences in amino acid sequences, which may affect the spatial structure and active site of the enzyme, thereby leading to different biological activities. Therefore, the actual function of isozymes must be combined with specific reactions, and further verified and judged by experiments.

[0027] Based on the problems existing in the prior art, the inventors have mined and screened more inositol-3-phosphate synthetases and inositol monophosphatases. The new multi-enzyme catalytic system can improve the conversion efficiency of starch, prepare inositol, and can be directly used by whole cells, the process is simple, and can be well applied to the in vitro enzyme catalytic conversion biosynthesis pathway of inositol, and is suitable for high substrate concentration feed production.

[0028] Example 1 Preparation of enzymes required for inositol biocatalytic synthesis The enzymes required for inositol synthesis include isoamylase (IA), α-glucan phosphorylase (αGP), phosphoglucomutase (PGM), inositol-3-phosphate synthase (IPS), and inositol monophosphatase (IMP). Among them, IA is used to hydrolyze α-1, 6 glycosidic bonds; αGP is used to hydrolyze α-1, 4 glycosidic bonds 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-3-phosphate; and IMP is used to dephosphorylate inositol-3-phosphate to generate inositol.

[0029] 1.1 Synthesis of recombinant plasmids For convenience of expression in the text, we respectively mark isoamylase, α-glucan phosphorylase, phosphoglucomutase, inositol-3-phosphate synthase, and inositol monophosphatase as A, B, C, D, and E. Isocenzymes from different biological sources are distinguished by adding numbers after the corresponding numbers. The numbers, species sources, and sequences of the related enzymes are shown in Table 2.

[0030] Table 2 Numbers, species sources, and sequences of enzymes

[0031]

[0032] Note: D0 and E0 are inositol-3-phosphate synthase and inositol monophosphatase disclosed in CN201510184621.4, respectively.

[0033] The nucleotide sequences shown in Table 2 are respectively inserted into the NcoI / XhoI enzyme cutting sites of pET28a plasmid, thereby obtaining different recombinant plasmid sequences.

[0034] Among them, part of the amino acid sequences and nucleotide sequences are as follows: SEQ ID NO:6 MVRVVILGQGYVASIFASGLEKIKAGKMEPYGVPLADELPIKIKDIEIVGSYDVDSNKVGKDLYEVVKSYDPDAPESLRGITIRKGVHLGSLRNLPLTPTGLDDEMTLKEAVDHLVNEWKELKPDVFVNVCTTEAFVPFESREELEKAIEENNKERLTATQFYVYAAAKYAKEVGGAAFVNAIPTLIANDPVFVELAKESNLVIFGDDGATGATPLTADILSHLAQRNRYVLDIAQFNIGGNQDFLALTDEERNRSKEFTKSSVVEDLLGYNAPHYIKPTGFLEPLGDKKFIAMHIEYVSFNGAHDELVITGRINDSPALAGLLVDLVRLGKIAIEKKEFGTVYEVNAFYMKNPGPKEARNIPRIIAHEKMRMWAGLKPRWL SEQ ID NO: 7 MVRVVIVGQGYVASIFAVGLERIKRGDLGYYGVPLANELPIKVEDIQIVGSYDVDKTKIGLPLYEIVQRYWKGDVPETLQEVFVRKGVHLRSLRNLPIEAVGLEDEMSLRDAIEHLVSEWKELKPDVIVNVPTTEAFVPFEDLEKLEKAIKEDDRDRLTATQVYAYAAAQYAKEVGGAAFVNAIPTLIANDPAFVELAKESNLVIFGDDGATGATPLTADVLAHLAQRNRHVRDIAQFNIGGNTDFLALTDKERNRSKEYTKSSVVEDILGYNAPHFIKPTGFLEPLGDKKFIAMHIEYVSFNGAIDELVITGRINDSPALAGLLVDLVRLGKIAVDRKEFGTVYEVNAFYMKNPGPKEAKNLPRIIAYEKMRQWAGLKPRYL SEQ ID NO: 8 MVKLVRVAIIGQGYVASIFAVGLERIKLGELGYYGVPLANELPIKVEDIQIVASYDVDKSKIGLPLSEIVKRYWNGHIPESLQEVYVRKGVHLRSLRNLPIEASGLEEEMTLKEAVEHLVNEWKESKVEVILNVPTTEAFTPFGKLEELEKAINENNKDRLTATQVYAYAAAQYAKEVGGAAFVNAIPTLIANDPAFVELAKESNLVIFGDDGATGATPLTADILGHLAQRNRHVLDIAQFNIGGNTDFLALTDKERNKSKEYTKSSVVEDILGYDAPHYIKPTGYLEPLGDKKFIAMHIEYVSFNGAHDELIITGRINDSPALAGLLVDLARLGKIAVEKKAFGTVYEVNAFYMKNPGPRDARNVPRIIAYEKLRQWVGLPPRYL SEQ ID NO: 12 MDERDALRISREIAGEVRKAIASMPLRERVKDVGMGKDGTPTKAADRVAEDAALEILRKERVTVVTEESGVLGEGDVFVALDPLDGTFNATRGIPVYSVSLCFSYSDKLKDAFFGYVYNLATGDEYYADSSGAYRNGERIEVSDAEELYCNAIIYYPDRKFPFKRMRIFGSAATELCFFADGSFDCFLDIRPGKMLRIYDAAAGVFIAEKAGGKVTELDGESLGNKKFDMQERLNIVAANEKLHPKLLELIK SEQ ID NO: 13 MEFNWSEIALNTAKELEEKIMPLFGTKKAGENVGTNVSGDVTKYVDKVAEDIILKRLVPLGVNVVSEEVGTVDSGSDYTVVVDPLDGSYNFSAGIPIFAFSLGIFKGKKPVYGAIYEFLPENFYEAKPGKGAYLNGERIRVNEPEPGKEALSFYTRGRCLGLVKKVKRVRVLGAIAVELAYVARGSLDGVFDIRNYVRPTDVAAGVLLVREAGGIVTDERGREFEVKLSATEKTNIIAVANERLLNTILEAMKDEP SEQ ID NO: 14 MDRMDFSIKIARKVGLYLMEHWGNAENVRQKSSFQDLVSDCDKQAQKMIVQKIKDHFPDDAILAEEGLFEKGDRMWIIDPIDGTMNYVHGLPSFAIGIAYVEKEQVILGVAHDPVLNETYYAIKGQGAYKNGERINVSENSLLKDSIGNTGFYTDFTGIFISAIEKKVRRVRMTGSAILAGAYVACGRFDFFIAKRANSWDVAPLFVLVPEAGGIVTDLSGNQAHLNTGNFLFSNGLLHDQVLEVIREVNKKVRK

[0035] 1.2 Construction of recombinant E. coli BL21(DE3) strain 1 μL of the constructed recombinant plasmid was added to 50 μL of E. coli BL21(DE3) competent cells, which were placed on ice for 30 min, then heat shocked at 42°C for 45 s and immediately placed on ice for 2 min. After adding 600 μL of LB medium without resistance, the cells were recovered at 37°C for 60 min. An appropriate amount of bacterial liquid was spread on LB plates containing kanamycin (kanamycin concentration 50 μg / mL), and incubated at 37°C overnight. A single colony was selected and inoculated in liquid LB medium containing kanamycin (50 μg / mL), and after several hours of culture, 1 mL of 50% glycerol was added to 1 mL of bacterial liquid when the OD600 was about 1-2. The recombinant strain was stored at -80°C.

[0036] 1.3 Preparation of primary purified enzyme solution, homogeneous enzyme solution and whole-cell enzyme The following is the preparation process of the enzymes required for inositol synthesis. The preparation was carried out in small volume, and the preparation in large volume can be carried out in proportion or according to the actual situation, which is not described here.

[0037] (1) Strain activation: pick the glycerol strain and inoculate in 5 mL of liquid LB medium (containing 50 μg / mL kanamycin), and incubate at 37°C with 220 rpm shaking for 4-5 h.

[0038] (2) The activated strain was inoculated into a flask containing 150 mL of TB medium at a seeding amount of 1% (v / v), and incubated at 37°C with 250 rpm shaking until the OD600 was about 0.6-1.2. Then, 0.1 mM IPTG was added, and the culture was incubated at 22°C with 150 rpm shaking overnight for induction expression of enzyme protein.

[0039] (3) After induction culture, the wet bacteria were collected by centrifugation, and the bacterial slurry was used as whole-cell enzyme.

[0040] (4) Resuspend the bacterial slurry in 100 mM potassium phosphate buffer (KPi buffer, pH 7.0) to a concentration of 300 g / L to obtain a resuspension. Homogenize the resuspension using a high-pressure homogenizer at a pressure of 800 MPa for 5-10 min until the resuspension becomes transparent, thereby obtaining a homogenized enzyme solution.

[0041] (5) Stir and heat the obtained homogenized enzyme solution in a water bath at a temperature of 70°C at a stirring speed of 200 rpm for 30 min, and then centrifuge at 10,000 rpm for 10 min. Discard the precipitate and retain the supernatant, thereby obtaining a primary purified enzyme solution from which most impurity proteins have been removed.

[0042] Example 2 Screening of myo-inositol monophosphatase under the condition of 2 mL reaction system and 10 g / L corn starch Prepare the primary purified enzyme solution according to the method described in 1.3 of Example 1. In a 2 mL reaction system, the components and their contents are as follows: 50 μL / mL of the primary purified enzyme solution of glucan phosphorylase, 50 μL / mL of the primary purified enzyme solution of phosphoglucomutase, 250 μL / mL of the primary purified enzyme solution of myo-inositol-3-phosphate synthase, 50 μL / mL of the primary purified enzyme solution of myo-inositol monophosphatase, 100 mM of potassium phosphate buffer (pH 7.0), 10 mM of magnesium chloride, and 10 g / L of corn starch. Perform catalytic reaction at 80°C for 20 hours. After the reaction, dilute the reaction solution and centrifuge at 12,000 rpm for 15 min. Take the supernatant and perform LC-MS detection. The LC-MS detection method, myo-inositol content calculation method, and reaction yield calculation method are as described above.

[0043] The catalytic reaction yields of the combinations of the final glucan phosphorylase B, phosphoglucomutase C, myo-inositol-3-phosphate synthase D0, and different myo-inositol monophosphatases are shown in Table 3. Myo-inositol monophosphatase E1 has relatively better catalytic activity, with a yield of 9.7%.

[0044] Table 3 Catalytic reaction yields of different combinations of myo-inositol monophosphatases under the condition of 2 mL reaction system and 10 g / L corn starch Catalytic reaction yields of different combinations of myo-inositol monophosphatases Reaction No. Glucan phosphorylase (50 μL / mL) Phosphoglucomutase Myo-inositol 3-phosphate synthase Myo-inositol monophosphatase Yield % 3-1 B C D0 E1 9.7 3-2 B C D0 E2 6.4 3-3 B C D0 E4 6.5 3-4 B C D0 E0 3.1 Example 3 Screening of myo-inositol-3-phosphate synthase under the condition of 2 mL reaction system and 10 g / L corn starch Homogeneous enzyme solution was prepared as described in 1.3 of Example 1. The components and their contents in 2 mL reaction system were as follows: 50 μL / mL of homogeneous enzyme solution of glucan phosphorylase, 50 μL / mL of homogeneous enzyme solution of phosphoglucomutase, 250 μL / mL of homogeneous enzyme solution of myo-inositol-3-phosphate synthase, 50 μL / mL of homogeneous enzyme solution of myo-inositol monophosphatase, 100 mM of potassium phosphate buffer (pH 7.0), 10 mM of magnesium chloride, 10 g / L of corn starch. Catalytic reaction was carried out at 80°C for 20 hours. After the reaction, the reaction solution was diluted and centrifuged at 12000 rpm for 15 min, and the supernatant was detected by LC-MS. The LC-MS detection method, myo-inositol content calculation method and reaction yield calculation method were as described above.

[0045] The catalytic reaction yields of the multi-enzyme combination of final glucan phosphorylase B, phosphoglucomutase C, myo-inositol monophosphatase E1 and different myo-inositol-3-phosphate synthases were shown in Table 4. Under the conditions of 2 mL reaction system and 10 g / L corn starch, the reaction yields of the multi-enzyme systems containing D9, D7 and D4 were higher, reaching 73%, 67.3% and 61.9%, respectively.

[0046] Table 4. Catalytic reaction yields of different myo-inositol-3-phosphate synthases under the conditions of 2 mL reaction system and 10 g / L corn starch Catalytic reaction yields of different myo-inositol-3-phosphate synthases Reaction No. Glucan phosphorylase Phosphoglucomutase Myo-inositol 3-phosphate synthase Myo-inositol monophosphatase Yield % 4-1 B C D9 E1 73.0 4-2 B C D7 E1 67.3 4-3 B C D4 E1 61.9 4-4 B C D11 E1 11.0 4-5 B C D0 E1 8.9 4-6 B C D10 E1 4.0 4-7 B C D1 E1 ND 4-8 B C D12 E1 ND ND: not detected Example 4. Catalytic reaction results of different myo-inositol-3-phosphate synthases under the conditions of 100 mL reaction system and 150 g / L corn starch According to the method described in 1.3 of Example 1, whole-cell enzymes in the form of bacterial slurry were prepared. The activity of the screened myo-inositol-3-phosphate synthases D4, D7 and D9 with higher activity was verified under the conditions of 100 mL reaction system and 150 g / L starch concentration. The components in 100 mL reaction system were as follows: 25 g / L of whole-cell enzyme of glucan phosphorylase in the form of bacterial slurry, 25 g / L of whole-cell enzyme of phosphoglucomutase in the form of bacterial slurry, 64 g / L of whole-cell enzyme of myo-inositol-3-phosphate synthase in the form of bacterial slurry, 25 g / L of whole-cell enzyme of myo-inositol monophosphatase in the form of bacterial slurry, 100 mM of potassium phosphate buffer (pH 7.0), 80 mM of magnesium chloride, 150 g / L of corn starch. Catalytic reaction was carried out at 80°C for 20 hours. After the reaction, the reaction solution was diluted and centrifuged at 12000 rpm for 15 min, and the supernatant was detected by LC-MS. The LC-MS detection method, myo-inositol content calculation method and reaction yield calculation method were as described above.

[0047] Finally, the catalytic reaction yield of the multi-enzyme combination of glucan phosphorylase B, phosphoglucomutase C, myo-inositol monophosphatase E1 and myo-inositol-3-phosphate synthase D4, D7 and D9 using slurry as whole-cell enzyme was shown in Table 5. The results showed that under the reaction conditions of 100 mL reaction system, 150 g / L corn starch and using whole-cell enzyme as raw material, the multi-enzyme system with myo-inositol-3-phosphate synthase D4 had better catalytic activity, and the yield was 68%, and the reaction yields of the multi-enzyme systems with D9 and D7 were 63.9% and 57.3%, respectively.

[0048] Table 5 Catalytic reaction yield of different combinations of myo-inositol-3-phosphate synthases under the conditions of 100 mL reaction system and 150 g / L corn starch Catalytic reaction yield of different combinations of myo-inositol-3-phosphate synthases Reaction No. Glucan phosphorylase Phosphoglucomutase Myo-inositol 3-phosphate synthase Myo-inositol monophosphatase Yield % 5-1 B C D4 E1 68.0 5-2 B C D7 E1 57.3 5-3 B C D9 E1 63.9 Example 5 Catalytic effect comparison of adding isoamylase in multi-enzyme system under the conditions of 100 mL reaction system and 150 g / L corn starch Starch generally contains a large number of branched chains, and glucan phosphorylase only acts on a-1, 4 glycosidic bonds, while the branched chains are connected to the main chain by a-1, 6 glycosidic bonds. In order to improve the utilization of starch, isoamylase (IA) can be added to hydrolyze a-1, 6 glycosidic bonds.

[0049] The whole-cell enzyme in the form of slurry was prepared according to the method described in 1.3 of Example 1. The 100 mL reaction system contained the following components: 25 g / L whole-cell enzyme of glucan phosphorylase in the form of slurry, 25 g / L whole-cell enzyme of phosphoglucomutase in the form of slurry, 64 g / L whole-cell enzyme of myo-inositol-3-phosphate synthase in the form of slurry, 25 g / L whole-cell enzyme of myo-inositol monophosphatase in the form of slurry, 100 mM potassium phosphate buffer (pH 7.0), 80 mM magnesium chloride, 150 g / L corn starch, and 5 g / L whole-cell enzyme of isoamylase in the form of slurry was added to the experimental group, and no isoamylase was added to the control group. The catalytic reaction was carried out at 80°C for 20 hours. After the reaction was completed, the reaction solution was diluted and centrifuged at 12000 rpm for 15 min, and the supernatant was detected by LC-MS. The LC-MS detection method, myo-inositol content calculation method and reaction yield calculation method are as described above.

[0050] The catalytic reaction yield comparison results of adding isoamylase A are shown in Table 6. The results showed that adding isoamylase in the multi-enzyme catalytic system can improve the catalytic efficiency.

[0051] Table 6 Catalytic reaction yield comparison results of adding isoamylase under the conditions of 100 mL reaction system and 150 g / L corn starch Catalytic reaction yield comparison results of adding isoamylase Reaction No. Isoamylase Glucan phosphorylase Phosphoglucomutase Myo-inositol 3-phosphate synthase Myo-inositol monophosphatase Yield % 6-1 / B C D4 E1 66.8 6-2 A B C D4 E1 71.0 Example 6 Optimization of reaction temperature under the condition of 100 mL reaction system and 150 g / L corn starch According to the method described in 1.3 of Example 1, the whole-cell enzyme in the form of bacterial slurry was prepared. The following ingredients were added to a 100 mL reaction system: 5 g / L of bacterial slurry form of isoamylase whole-cell enzyme, 25 g / L of bacterial slurry form of glucan phosphorylase whole-cell enzyme, 25 g / L of bacterial slurry form of phosphoglucomutase whole-cell enzyme, 64 g / L of bacterial slurry form of myo-inositol-3-phosphate synthase whole-cell enzyme, 25 g / L of bacterial slurry form of myo-inositol monophosphatase whole-cell enzyme, 100 mM of potassium phosphate buffer (pH 7.0), 80 mM of magnesium chloride, and 150 g / L of corn starch. Catalytic reactions were carried out at 60°C, 70°C, 75°C, 80°C, 85°C, and 90°C, respectively, for 20 hours. After the reaction was completed, the reaction solution was diluted, centrifuged at 12,000 rpm for 15 min, and the supernatant was subjected to LC-MS detection. The LC-MS detection method, myo-inositol content calculation method, and reaction yield calculation method are as described above.

[0052] Finally, the multi-enzyme reaction system composed of isoamylase A, glucan phosphorylase B, phosphoglucomutase C, myo-inositol monophosphatase E1, and myo-inositol-3-phosphate synthase D4 had the highest catalytic reaction yield when the reaction temperature was controlled at 80°C. The results are shown in Table 7.

[0053] Table 7 Catalytic reaction yield at different reaction temperatures Reaction No. Isoamylase Glucan phosphorylase Phosphoglucomutase Myo-inositol 3-phosphate synthase Myo-inositol monophosphatase Reaction temperature Yield % 7-1 A B C D4 E1 60℃ 17.5 7-2 A B C D4 E1 70℃ 27.0 7-3 A B C D4 E1 75℃ 69.6 7-4 A B C D4 E1 80℃ 72.0 7-5 A B C D4 E1 85℃ 57.6 7-6 A B C D4 E1 90℃ 60.0 Example 7 Optimization of reaction time under the condition of 100 mL reaction system and 150 g / L corn starch According to the method described in 1.3 of Example 1, the whole-cell enzyme in the form of bacterial slurry was prepared. The following ingredients were added to a 100 mL reaction system: 25 g / L of bacterial slurry form of glucan phosphorylase whole-cell enzyme, 25 g / L of bacterial slurry form of phosphoglucomutase whole-cell enzyme, 64 g / L of bacterial slurry form of myo-inositol-3-phosphate synthase whole-cell enzyme, 25 g / L of bacterial slurry form of myo-inositol monophosphatase whole-cell enzyme, 100 mM of potassium phosphate buffer (pH 7.0), 80 mM of magnesium chloride, 150 g / L of corn starch, and 5 g / L of bacterial slurry form of isoamylase whole-cell enzyme. Catalytic reactions were carried out at 80°C for 10, 20, 40, 60, 80, and 120 hours, respectively. After the reaction was completed, the reaction solution was diluted, centrifuged at 12,000 rpm for 15 min, and the supernatant was subjected to LC-MS detection. The LC-MS detection method, myo-inositol content calculation method, and reaction yield calculation method are as described above.

[0054] Finally, the multi-enzyme reaction system composed of isoamylase A, glucan phosphorylase B, phosphoglucomutase C, myo-inositol monophosphatase E1 and myo-inositol-3-phosphate synthase D4, the reaction time is within 20-80 hours, and the reaction yield is more than 70%. The yield decreases when the reaction is continued, and the results are shown in Table 8.

[0055] Table 8 Catalytic reaction yield under different reaction time Reaction No. Isoamylase Glucan phosphorylase Phosphoglucomutase Myo-inositol 3-phosphate synthase Myo-inositol monophosphatase Reaction time (hours) Yield % 8-1 A B C D4 E1 10 65.0 8-2 A B C D4 E1 20 74.0 8-3 A B C D4 E1 40 78.0 8-4 A B C D4 E1 60 76.0 8-5 A B C D4 E1 80 72.0 8-6 A B C D4 E1 120 69.0 Example 8 Preparation of myo-inositol by multi-enzyme catalysis under the condition of 150 g / L corn starch in 1L reaction system The whole-cell enzyme in the form of bacteria slurry was prepared according to the method described in 1.3 of Example 1. The following ingredients were added in a 1L reaction system: 25 g / L bacteria slurry form of glucan phosphorylase whole-cell enzyme, 25 g / L bacteria slurry form of phosphoglucomutase whole-cell enzyme, 64 g / L bacteria slurry form of myo-inositol-3-phosphate synthase whole-cell enzyme, 25 g / L bacteria slurry form of myo-inositol monophosphatase whole-cell enzyme, 100 mM potassium phosphate buffer (pH 7.0), 80 mM magnesium chloride, 150 g / L corn starch, and 5 g / L bacteria slurry form of isoamylase whole-cell enzyme.

[0056] The catalytic reaction was carried out at 80°C for 40 hours. After the reaction was completed, the reaction solution was diluted and centrifuged at 12000 rpm for 15 min, and the supernatant was detected by LC-MS. The LC-MS detection method, myo-inositol content calculation method and reaction yield calculation method are as described above.

[0057] Finally, the catalytic reaction yield of isoamylase A, glucan phosphorylase B, phosphoglucomutase C, myo-inositol monophosphatase E1 and myo-inositol-3-phosphate synthase D4, D7 and D9 in a 1L reaction system under the condition of 40 hours of reaction time is shown in Table 9. The reaction yield of the multi-enzyme combination of D4 under this condition can reach 85%, and the reaction yields of the multi-enzyme combinations of D7 and D9 are 76% and 81% respectively, indicating that the multi-enzyme system composed of A-B-C-D4-E1 has excellent potential for industrial application.

[0058] Table 9 Multi-enzyme catalytic synthesis of myo-inositol under the condition of 150 g / L corn starch in 1L system Reaction No. Isoamylase Glucan phosphorylase Phosphoglucomutase Myo-inositol 3-phosphate synthase Myo-inositol monophosphatase Reaction yield % 9-1 A B C D4 E1 85.0 9-2 A B C D7 E1 76.0 9-3 A B C D9 E1 81.0 Example 9 Preparation of myo-inositol by multi-enzyme catalysis under different starch concentrations in 1L reaction system The whole-cell enzyme in the form of bacterial slurry was prepared according to the method described in 1.3 of Example 1. In a 1 L reaction system, the following ingredients were added: 25 g / L of the whole-cell enzyme of phosphoglucomutase in the form of bacterial slurry, 25 g / L of the whole-cell enzyme of phosphoglucomutase in the form of bacterial slurry, 64 g / L of the whole-cell enzyme of myo-inositol-3-phosphate synthase in the form of bacterial slurry, 25 g / L of the whole-cell enzyme of myo-inositol monophosphatase in the form of bacterial slurry, 100 mM of potassium phosphate buffer (pH 7.0), 80 mM of magnesium chloride, 5 g / L of the whole-cell enzyme of isoamylase in the form of bacterial slurry, 150 g / L, 200 g / L and 250 g / L of corn starch, respectively. The catalytic reaction was carried out at 80°C for 40 hours. After the reaction was completed, the reaction solution was diluted and centrifuged at 12000 rpm for 15 min, and the supernatant was detected by LC-MS. The LC-MS detection method, the myo-inositol content calculation method and the reaction yield calculation method are as described above.

[0059] Finally, the catalytic reaction yield of the multi-enzyme system composed of isoamylase A, phosphoglucomutase B, phosphoglucomutase C, myo-inositol monophosphatase E1 and myo-inositol-3-phosphate synthase D4 under different starch concentrations is shown in Table 11: under the concentration of 150 g / L of corn starch, the measured myo-inositol content reached 127.5 g / L, the reaction yield reached 85%, and the LC-MS detection result is shown in Figure 2 under the concentration of 200 g / L of corn starch, the measured myo-inositol content reached 150 g / L, the reaction yield reached 75%; under the concentration of 250 g / L of corn starch, the measured myo-inositol content reached 165 g / L, the reaction yield reached 66%, and the results showed that under the condition of high concentration of starch, the multi-enzyme catalytic system could still efficiently catalyze the synthesis of myo-inositol.

[0060] Table 10 Catalytic reaction yield under different starch concentrations in a 1 L system Reaction No. Isoamylase Glucan phosphorylase Phosphoglucomutase Myo-inositol 3-phosphate synthase Myo-inositol monophosphatase Starch concentration Reaction yield % 10-1 A B C D4 E1 150 g / L 85.0 10-2 A B C D4 E1 200 g / L 75.0 10-3 A B C D4 E1 250 g / L 66.0 Example 10 Preparation of myo-inositol by the combination of myo-inositol-3-phosphate synthase D4 and myo-inositol monophosphatase E1 under the condition of 200 g / L of corn starch in a 60 L reaction system The whole-cell enzyme in the form of bacterial slurry was prepared according to the method described in 1.3 of Example 1. The following ingredients were added in a stainless steel reactor: 25 g / L of the whole-cell enzyme of glucan phosphorylase in the form of bacterial slurry, 25 g / L of the whole-cell enzyme of phosphoglucomutase in the form of bacterial slurry, 64 g / L of the whole-cell enzyme of myo-inositol-3-phosphate synthase in the form of bacterial slurry, 25 g / L of the whole-cell enzyme of myo-inositol monophosphatase in the form of bacterial slurry, 100 mM of potassium phosphate buffer (pH 7.0), 80 mM of magnesium chloride, 200 g / L of corn starch, 5 g / L of the whole-cell enzyme of isoamylase in the form of bacterial slurry, and the final reaction system was 60 L. The catalytic reaction was carried out at 80°C for 42 hours. After the reaction was completed, the reaction solution was diluted and centrifuged at 12,000 rpm for 15 min, and the supernatant was detected by LC-MS. The LC-MS detection method, the myo-inositol content calculation method and the reaction yield calculation method are as described above.

[0061] Under the conditions of a final reaction system of 60 L and a reaction time of 42 hours, the multi-enzyme combination of isoamylase A, glucan phosphorylase B, phosphoglucomutase C, myo-inositol-3-phosphate synthase D4 and myo-inositol monophosphatase E1 catalyzed 200 g / L of corn starch to produce myo-inositol, and the unit yield reached 161 g / L, and the reaction yield reached 80.5%, indicating that the multi-enzyme combination has good industrial application prospects.

Claims

1. Use of a myo-inositol monophosphatase in an enzyme-catalyzed reaction system for the preparation of myo-inositol, wherein, The myo-inositol monophosphatase is derived from Archaeoglobus fulgidus, and the amino acid sequence is shown as SEQ ID NO: 12; or the myo-inositol monophosphatase is derived from Thermococcus kodakarensis, and the amino acid sequence is shown as SEQ ID NO: 13; or the myo-inositol monophosphatase is derived from Pseudothermotoga lettingae, and the amino acid sequence is shown as SEQ ID NO:

14.

2. A method of preparing myo-inositol, characterized by: It is to add glucan phosphorylase, phosphoglucomutase, myo-inositol-3-phosphate synthase and myo-inositol monophosphatase as the enzyme catalysts for catalyzing the substrate to generate myo-inositol; the myo-inositol monophosphatase is derived from Archaeoglobus fulgidus, and the amino acid sequence is shown as SEQ ID NO: 12; or the myo-inositol monophosphatase is derived from Thermococcus kodakarensis, and the amino acid sequence is shown as SEQ ID NO: 13; or the myo-inositol monophosphatase is derived from Pseudothermotoga lettingae, and the amino acid sequence is shown as SEQ ID NO:

14.

3. The method of claim 2, wherein: The myo-inositol monophosphatase is derived from Archaeoglobus fulgidus, and the amino acid sequence is shown as SEQ ID NO:

12.

4. The method of claim 2, wherein: The myo-inositol-3-phosphate synthase is derived from Thermococcus kodakarensis, and the amino acid sequence is shown as SEQ ID NO:

6.

5. The method of claim 2, wherein: The myo-inositol-3-phosphate synthase is derived from Pyrococcus yayanosii, and the amino acid sequence is shown as SEQ ID NO:

7.

6. The method of claim 2, wherein: The myo-inositol-3-phosphate synthase is derived from Pyrococcus abyssi, and the amino acid sequence is shown as SEQ ID NO:

8.

7. The process for the preparation of inositol according to any one of claims 2-6, characterized in that: The enzyme catalysts further include isoamylase.

8. The process for the preparation of inositol according to any one of claims 2-6, characterized in that: It comprises the following steps: a. weighing starch, adding glucan phosphorylase, phosphoglucomutase, myo-inositol-3-phosphate synthase and myo-inositol monophosphatase; b. performing catalytic reaction at 60-90℃ for 10-120 hours, and obtaining the product.

9. The preparation method of myo-inositol according to claim 8, characterized in that: the amount of starch in the reaction system in step a is 10g / L to 250g / L; the enzyme catalytic reaction temperature in step b is 75-80℃, preferably, the enzyme catalytic reaction temperature is 80℃; and the reaction time is 20-80 hours.

10. The process for the preparation of inositol according to any one of claims 2-6, characterized in that: The myo-inositol-3-phosphate synthase and the myo-inositol monophosphatase are in the form of primary purified enzyme liquid, homogeneous enzyme liquid or whole cell enzyme.

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