A myo-inositol-3-phosphate synthase mutant and application thereof, and a preparation method of myo-inositol

Abstract

The application provides a myo-inositol-3-phosphate synthase mutant, which is obtained by mutating a myo-inositol-3-phosphate synthase with an amino acid sequence as shown in SEQ ID NO: 4 at positions 55, 57 and 273 respectively. The application also provides a preparation method of myo-inositol. The inventors have screened a wild-type myo-inositol-3-phosphate synthase and a myo-inositol monophosphatase with good performance, which can be used in cooperation with a glucan phosphorylase and a glucose phosphate mutase to catalyze starch to generate myo-inositol more efficiently. The myo-inositol-3-phosphate synthase mutant is applied to in-vitro non-fermentation biosynthesis of myo-inositol, combined with myo-inositol monophosphatases E1 and E5, and in a way of feeding whole cells, so that starch can be converted into myo-inositol in a one-pot method, without using NAD + coenzymes, and has a good industrial application prospect.

Description

Technical Field

[0001] This invention relates to an inositol-3-phosphate synthase mutant and its application, as well as a method for preparing inositol, belonging to the field of enzyme engineering. Background Technology

[0002] Inositol (MI), also known as cyclohexanol, is a direct precursor of glucuronic acid and is essential for the growth of humans, animals, and microorganisms. As a nutritional supplement, inositol is widely used in the pharmaceutical, food, and animal feed industries. In recent years, with the continuous development of downstream markets, the demand for inositol has shown a year-on-year upward trend.

[0003] Currently, the main production processes for inositol include pressurized hydrolysis, atmospheric pressure hydrolysis, enzymatic hydrolysis, microbial fermentation, and in vitro enzyme synthesis. Among these, microbial fermentation for inositol production is based on theoretical research into the inositol metabolic pathway in yeast. The Shirai Makoto team in Japan and Pramod Agmwal et al. in the United States have made significant contributions to this field. During the 1990s, the Shirai Makoto team had eight patents related to microbial fermentation for inositol production filed with the European Patent Office.

[0004] In vitro enzymatic synthesis is based on research into the inositol metabolic pathway and involves the stepwise or one-step synthesis of inositol through in vitro multi-enzyme catalysis. While in vitro enzymatic synthesis offers advantages such as environmentally friendly production processes and high inositol yields, existing processes suffer from varying degrees of enzyme activity instability, high costs, low raw material utilization, and low yields, thus exhibiting significant limitations.

[0005] PCT application WO2014197702A1 discloses a reaction for producing glucose-6-phosphate (G6P) from starch or maltodextrin using α-glucan phosphorylase and glucose phosphate mutase. Todd B. Reynolds et al., Microbiology (2009), 155, 1386–1396, disclose an in vitro production method that converts glucose-6-phosphate (G6P) to inositol-3-phosphate via inositol-3-phosphate synthase, followed by conversion of inositol-3-phosphate to inositol via inositol monophosphatase.

[0006] Patent document CN106148425A discloses a method for producing inositol from starch and starch derivatives via a one-step in vitro multi-enzyme catalysis. However, the starch used in this method is soluble starch, and the substrate concentration is very low. The separation and purification steps of the enzymes in the multi-enzyme system are cumbersome. 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 that achieves a one-step conversion of starch to inositol. All enzymes in this method are derived from hyperthermophilic microorganisms and can be expressed through high-density fermentation of E. coli. However, simple purification using heat precipitation is required, and the substrate concentration is only up to 125 g / L after scaling up the reaction, resulting in a low yield of inositol per unit volume.

[0007] Patent document CN118028276A also discloses an inositol-3-phosphate synthase mutant, its preparation method, applications, and a method for preparing inositol. This method involves enzymatically reacting maltodextrin with the inositol-3-phosphate synthase mutant, inositol monophosphatase, and optionally maltodextrin phosphorylase and glucose-6-phosphate mutase to obtain inositol. However, this method is only at the laboratory-scale level and also suffers from low raw material utilization.

[0008] In summary, existing in vitro enzymatic conversion methods for preparing inositol utilize enzymes with limited selectivity and are mostly limited to small-scale laboratory applications, thus significantly restricting their use in industrial production. Therefore, there is an urgent need to develop a new method for inositol production that can be used throughout the entire cell, offers high performance and yield, has a simple production process, low cost, is suitable for high substrate concentrations and high inositol production, and is easily scalable for large-scale production. Summary of the Invention

[0009] Existing methods for preparing inositol via in vitro multi-enzyme catalytic reactions have limitations for industrial production, such as cumbersome enzyme purification steps, complex production processes, and the problem of low-concentration substrate feeding. Furthermore, the enzymes used in enzyme conversion methods have limited selectivity and are not suitable for the preparation and application of inositol.

[0010] Based on the existing problems, the inventors screened out more inositol-3-phosphate synthase and inositol monophosphatase to form a new multi-enzyme reaction system, which can improve the conversion efficiency of starch and produce inositol.

[0011] Furthermore, this invention also provides an inositol-3-phosphate synthase mutant. By optimizing the binding and catalytic domains of wild-type inositol-3-phosphate synthase, the resulting mutant exhibits higher catalytic activity compared to the wild-type inositol-3-phosphate synthase. It can be used directly in whole cells, has a simple process, and can be well applied to the in vitro enzymatic conversion biosynthetic pathway of inositol. It is suitable for production with high substrate concentrations.

[0012] This invention provides a mutant of inositol-3-phosphate synthase, which is obtained by using inositol-3-phosphate synthase with the amino acid sequence shown in SEQ ID NO:4 as the parent, and by mutating aspartic acid at position 55 to glutamic acid to obtain mutant D4-D55E; mutating asparagine at position 57 to serine to obtain mutant D4-N57S; and mutating alanine at position 273 to arginine to obtain mutant D4-A273R.

[0013] Preferably, it uses mutant D4-D55E as the parent, and mutates asparagine to serine at position 57 to obtain mutant D4-D55E / N57S.

[0014] More preferably, it uses D4-D55E / N57S as the parent, and mutates the alanine at position 273 to threonine, proline, asparagine, and valine, respectively, to obtain mutants: D4-D55E / N57S / A273T, D4-D55E / N57S / A273P, D4-D55E / N57S / A273N, and D4-D55E / N57S / A273V.

[0015] The mutant is D4-D55E / N57S / A273P, and its amino acid sequence is shown in SEQ ID NO:19.

[0016] This invention provides the application of the aforementioned inositol-3-phosphate synthase mutant in the synthesis of inositol.

[0017] This invention provides a method for preparing inositol, which uses starch as a substrate and adds glucan phosphorylase, glucose phosphate mutase, inositol-3-phosphate synthase or a mutant of the aforementioned inositol-3-phosphate synthase, and inositol monophosphatase as enzyme catalysts to catalyze the production of inositol from the substrate; wherein the inositol-3-phosphate synthase is derived from Thermococcus kodakarensis, and its amino acid sequence is shown in SEQ ID NO:4; the inositol monophosphatase is derived from Archaeoglobus fulgidus, and its amino acid sequence is shown in SEQ ID NO:5; or, the inositol monophosphatase is derived from Thermotoga neapolitana, and its amino acid sequence is shown in SEQ ID NO:8.

[0018] The enzyme catalyst also includes isoamylase.

[0019] The preparation method of this invention includes the following steps:

[0020] a. Weigh out starch and add glucan phosphorylase, glucose phosphorylation mutase, inositol-3-phosphate synthase, and inositol monophosphatase.

[0021] b. Catalyze the reaction at 60-90℃ for 10-120 hours to obtain the final product.

[0022] In step a, the amount of starch used in the reaction system is 10 g / L to 150 g / L;

[0023] Preferably, the enzyme-catalyzed reaction temperature in step b is 80°C, and the reaction time is 20 hours.

[0024] The inositol-3-phosphate synthase and inositol monophosphatase mentioned herein are in the form of primary purified enzyme solution, homogenized enzyme solution or whole cell enzyme.

[0025] The beneficial effects of this invention are:

[0026] The inventors screened and obtained high-performance wild-type inositol-3-phosphate synthase and inositol monophosphatase, which can synergistically catalyze the more efficient conversion of starch to inositol with dextran phosphorylase and glucose phosphate mutase. Furthermore, the inventors modified wild-type inositol-3-phosphate synthase D4 (as shown in SEQ ID NO:4) as the parent, obtaining a superior mutant with significantly enhanced substrate binding capacity and catalytic activity compared to the wild-type inositol-3-phosphate synthase. This inositol-3-phosphate synthase mutant was applied to the in vitro non-fermentation biosynthesis of inositol, combined with inositol monophosphatases E1 and E5, using a whole-cell feeding method, enabling a one-pot, highly efficient conversion of high-concentration starch to inositol without the need for NAD+. + Coenzymes, such as these, have promising prospects for industrial applications. Attached Figure Description

[0027] Figure 1 This describes the catalytic conversion pathway for preparing inositol from starch using a multi-enzyme system.

[0028] Figure 2 The relative activity heatmaps for the initial screening of inositol-3-phosphate synthase mutant sites (where (a)-(h) are the relative activity heatmaps for the initial screening of sites K257, D208, Y70, L97, N55, N57, A273, and Q243, respectively, and positions B2, C3, and D4 of each heatmap represent the wild-type parent).

[0029] Figure 3 The LC-MS results of the preparation of inositol by a multi-enzyme system consisting of isoamylase A, dextran phosphorylase B, glucose phosphate mutase C, inositol monophosphatase E1 and inositol-3-phosphate synthase mutant D4-D55E / N57S / A273P were obtained in a 1L reaction system with 150 g / L corn starch.

[0030] Figure 4 The LC-MS results of the preparation of inositol were obtained by a multi-enzyme system consisting of isoamylase A, dextran phosphorylase B, glucose phosphate mutase C, inositol monophosphatase E5, and inositol-3-phosphate synthase mutant D4-D55E / N57S / A273P, under the conditions of 1L reaction system and 150 g / L corn starch. Detailed Implementation

[0031] The embodiments of the present invention are described in detail below. These embodiments are intended to explain the present invention and should not be construed as limiting the present invention.

[0032] Where specific techniques or conditions are not specified in the embodiments, they shall be performed in accordance with the techniques or conditions described in the literature in this field or according to the product instructions. All materials and reagents used in the embodiments are commonly used in this field and can be obtained through conventional commercial channels. The main materials and reagents are shown in Table 1.

[0033] Table 1 List of Main Materials and Reagents

[0034] name supplier Product Number Escherichia coli BL21(DE3) Beijing Qingke Biotechnology Co., Ltd. DZC201-96B100 pET28a plasmid Wuhan Miaoling Biotechnology Co., Ltd. P0023 Inositol Standard Shanghai McLean Biochemical Technology Co., Ltd. I811835 corn starch Shanghai McLean Biochemical Technology Co., Ltd. S818265 <![CDATA[High Fidelity DNA Polymerase KOD One TM PCR Master Mix]]> TOYOBO KMM-201

[0035] In the implementation of this invention, Escherichia coli can be cultured in shake flasks using conventional LB or TB media, or it can be cultured on a large scale in fermentation equipment using the cheaper corn steep liquor medium. The formulations of TB and LB media are as follows:

[0036] TB culture medium formula: tryptone 12 g / L; yeast extract 24 g / L; glycerol 4 mL / L; dipotassium hydrogen phosphate 16.43 g / L; potassium dihydrogen phosphate 2.31 g / L.

[0037] LB medium formula: peptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L.

[0038] The detection method using liquid chromatography-mass spectrometry (LC-MS) is as follows:

[0039] Inositol was detected using liquid chromatography-mass spectrometry (LC-MS). The chromatographic column was a phenomenex Gemini® 5 μm C18 column; the mobile phase was acetonitrile:water = 95:5; the flow rate was 1 mL / min; the column temperature was 35℃; and the injection volume was 1 μL. Inositol standards were dissolved in water to prepare standard solutions of different concentrations. The retention time of the inositol standard was approximately 2.689 min. The characteristic ion of inositol (m / z = +181.1) was extracted in SIM mode (Selected Ion Monitoring). The Mass response peak area of ​​this ion was used to plot a concentration-peak area curve, and the concentration of inositol in the sample was calculated based on this standard curve.

[0040] The relative activity calculation method is as follows:

[0041] Relative activity = peak area of ​​mutant enzyme catalysis / peak area of ​​parental enzyme catalysis × 100%, where the peak area of ​​parental enzyme catalysis is the average of three parental replicates.

[0042] The yield is calculated as follows:

[0043] After the catalytic reaction is completed, the concentration of inositol in the reaction solution is calculated according to the above method, and the reaction yield is calculated as (inositol concentration × reaction system volume) / starch feed amount × 100%.

[0044] The biosynthetic pathway for preparing inositol from starch as a substrate via in vitro multi-enzyme catalytic conversion is as follows: Figure 1 As shown, the last two enzymatic catalytic reactions are irreversible and have a crucial impact on the overall reaction. Furthermore, isoenzyme labeling in databases does not equate to identical biological activity. Even with the same functional annotation, differences in amino acid sequences between enzymes from different sources can affect their spatial structure and active sites, leading to variations in biological activity. Therefore, the actual function of isoenzymes must be verified and determined experimentally in conjunction with specific reactions.

[0045] Based on the problems existing in the current technology, the inventors discovered and screened high-performance wild-type inositol-3-phosphate synthase and inositol monophosphatase, which can synergistically catalyze starch to produce inositol more efficiently with dextran phosphorylase and glucose phosphate mutase.

[0046] Example 1: Preparation of relevant wild-type enzymes in a multi-enzyme system

[0047] The enzymes required for inositol synthesis include isoamylase (IA), α-glucanphosphorylase (αGP), glucose-phosphoglucomutase (PGM), inositol-3-phosphate synthase (IPS), and inositol monophosphatase (IMP). Specifically, IA hydrolyzes α-1,6-glycosidic bonds; αGP hydrolyzes α-1,4-glycosidic bonds to release glucose-1-phosphate; PGM converts glucose-1-phosphate to glucose-6-phosphate; IPS converts glucose-6-phosphate to inositol-3-phosphate; and IMP dephosphorylates inositol-3-phosphate to inositol.

[0048] 1.1 Synthesis of Recombinant Plasmids

[0049] For ease of description in this article, we have labeled isoamylase, α-glucan phosphorylase, glucose-phosphomutase, inositol-3-phosphate synthase, and inositol monophosphatase as A, B, C, D, and E, respectively. Isoenzymes from different biological sources are distinguished by adding numbers after their corresponding designations. The related enzyme designations, species origins, and sequences are shown in Table 2.

[0050] Table 2 Enzyme ID, Species Origin, and Sequence

[0051]

[0052] Note: D4 is the inositol-3-phosphate synthase disclosed in CN 119842829 A.

[0053] The nucleotide sequences shown in Table 2 were inserted between the NcoI / XhoI restriction sites of the pET28a plasmid to obtain different recombinant plasmid sequences.

[0054] Partial amino acid sequence is as follows:

[0055] SEQ ID NO:4:

[0056] MVRVVILGQGYVASIFASGLEKIKAGKMEPYGVPLADELPIKIKDIEIVGSYDVDSNKVGKDLYEVVKSYDPDAPESLRGITIRKGVHLGSLRNLPLTPTGLDDEMTLKEAVDHLVNEWKELKPDVFVVNVCTTEAFVPFESREELEKAIENNKERLTATQFYVYAAAKYAKEVGGAAFVNAIPTLIANDPVFVELAKESNLVIFGDDGATGATPLTADILSHLAQRNRYVLDIAQFNIGGNQDFLALTDEERNRSKEFTKSSVVEDLLGYNAPHYIKPTGFLEPLGDKKFIAMHIEYVSFNGAHDELVITGRINDSPALAGLLVDLVRLGKIAIEKKEFGTVYEVNAFYMKNPGPKEARNIPRIIAHEKMRMWAGLKPRWL

[0057] SEQ ID NO:5:

[0058] MDERDALRISREIAGEVRKAIASMPLRERVKDVGMGKDGTPTKAADRVAEDAALEILRKERVTVVTEESGVLGEGDVFVALDPLDGTFNATRGIPVYSVSLCFSYSDKLKDAFFGYVYNLATGDEYYADSSGAYRNGERIEVSDAEELYCNAIIYYPDRKFPFKRMRIFGSAATELCFFADGSFDCFLDIRPGKMLRIYDAAAGVFIAEKAGGKVTELDGESLGNKKFDMQERLNIVAANEKLHPKLLELIK

[0059] SEQ ID NO:8:

[0060] MEGGIELDRLDFSIKLLRRVGHFLMLHWGKVDSVEKKTGFKDIVTEIDKKAQEMIVEEIRKVFPDENIIAEEGISENGKKLWIIDPIDGTINFVHGLPNFSISIAYVENGEVKMGVVHAPALNETLYAEENGGAFLNGERIRVSGNTSLEECVGSTGSYVDFTGKFIEKMEKKTRRVRILGSAALNACYVGAGRVDFFVTWRINPWDIAAGLIVVKEAGGTVTDFAGKEANVFSKNFVFSNGLVHEEVLEVVNEVLKEIGEGK

[0061] SEQ ID NO:19:

[0062] MVRVVILGQGYVASIFASGLEKIKAGKMEPYGVPLADELPIKIKDIEIVGSYDVESSKVGKDLYEVVKSYDPDAPESLRGITIRKGVHLGSLRNLPLTPTGLDDEMTLKEAVDHLVNEWKELKPDVFVNVCTTEAFVPFESREELEKAIEENNKERLTATQFYVYAAAKYAKEVGGAAFVNAIPTLIANDPVFVELAKESNLVIFGDDGATGATPLTADILSHLAQRNRYVLDIAQFNIGGNQDFLALTDEERNRSKEFTKSSVVEDLLGYNPPHYIKPTGFLEPLGDKKFIAMHIEYVSFNGAHDELVITGRINDSPALAGLLVDLVRLGKIAIEKKEFGTVYEVNAFYMKNPGPKEARNIPRIIAHEKMRMWAGLKPRWL

[0063] 1.2 Construction of recombinant Escherichia coli BL21(DE3) strain

[0064] 1 μL of the constructed recombinant plasmid was added to 50 μL of *E. coli* BL21(DE3) competent cells and placed on ice for 30 min. Then, the cells were heat-shocked at 42℃ for 45 s and immediately placed on ice for 2 min. 600 μL of antibiotic-free LB medium was added, and the cells were incubated at 37℃ in a shaker for 60 min. An appropriate amount of the bacterial suspension was spread onto LB agar plates containing kanamycin resistance (50 μg / mL kanamycin) and incubated upside down overnight at 37℃. Single colonies were picked and transferred to kanamycin-resistant liquid LB medium (50 μg / mL kanamycin). After several hours of incubation until OD600 ≈ 1–2, 1 mL of 50% glycerol was added per 1 mL of bacterial suspension, and the recombinant strain was preserved at -80℃.

[0065] 1.3 Preparation of primary purified enzyme solution, homogenized enzyme solution and whole-cell enzyme

[0066] The following are the preparation processes of the enzymes required for inositol synthesis. All of these processes are carried out on a small-scale basis. Accordingly, the preparation can be carried out on a larger scale according to the proportions or actual conditions.

[0067] (1) Activation of bacterial strain: Pick glycerol bacterial strains and inoculate them into 5 mL of liquid LB medium (containing 50 μg / mL kanamycin), and culture at 37℃ with shaking at 220 rpm for 4-5 h.

[0068] (2) The activated strain was inoculated into a shake flask containing 150 mL of TB medium at an inoculation rate of 1% (v / v). The culture was carried out at 37°C and 250 rpm until OD600≈0.6~1.2. Then, IPTG was added to a final concentration of 0.1 mM and the culture was carried out overnight at 22°C and 150 rpm to induce the expression of the enzyme protein.

[0069] (3) After the induction culture is completed, the wet bacterial cells are collected by centrifugation, and the bacterial sludge is used as whole cell enzyme.

[0070] (4) Resuspend the bacterial sludge in 100 mM potassium phosphate buffer (KPi buffer, pH 7.0) to a concentration of 300 g / L to obtain a resuspension. Use a high-pressure homogenizer to homogenize and break down the resuspension at a pressure of 800 MPa for 5–10 min until the resuspension becomes clear to obtain the homogenized enzyme solution.

[0071] (5) The homogenized enzyme solution was heated in a water bath at 70°C for 200 rpm for 30 min. Then it was centrifuged at 10,000 rpm for 10 min. The precipitate was discarded and the supernatant was retained to obtain the primary purified enzyme solution with most of the impurity proteins removed.

[0072] Example 2: Screening of inositol monophosphatase in a 2 mL reaction system with 10 g / L corn starch.

[0073] The primary purified enzyme solution was prepared according to the method described in 1.3 of Example 1. The components and concentrations in a 2 mL reaction system were as follows: 50 μL / mL of primary purified dextran phosphorylase B, 50 μL / mL of primary purified glucose phosphate mutase C, 250 μL / mL of primary purified inositol-3-phosphate synthase D4, 50 μL / mL of primary purified inositol monophosphatases, 100 mM potassium phosphate buffer (pH 7.0), 10 mM magnesium chloride, and 10 g / L corn starch. The 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. The supernatant was analyzed by LC-MS. The LC-MS detection method, inositol content calculation method, and reaction yield calculation method were as described above.

[0074] Table 3 shows the catalytic yields of various combinations of glucan phosphorylase B, glucose phosphate mutase C, and inositol-3-phosphate synthase D4 with different inositol monophosphatases under a 2 mL reaction system and 10 g / L corn starch conditions. Among them, the multi-enzyme combination containing inositol monophosphatases E1 and E5 showed relatively better catalytic activity, with reaction yields of 62.5% and 55.6%, respectively.

[0075] Table 3. Yields of catalytic reactions of different inositol monophosphatase combinations under the condition of 2 mL reaction system and 10 g / L corn starch.

[0076] Numbering enzymes Dosage of dextran phosphorylase: 50 μL / mL Dosage of glucose phosphomutase: 50 μL / mL Dosage of inositol 3-phosphate synthase: 250 μL / mL Dosage of inositol monophosphatase: 50 μL / mL Yield % 3-1 B C D4 E0 18.3 3-2 B C D4 E1 62.5 3-3 B C D4 E2 ND 3-4 B C D4 E4 5.9 3-5 B C D4 E5 55.6

[0077] ND: Not detected

[0078] Example 3: Construction of an inositol-3-phosphate synthase D4 mutant library and initial activity screening in a 2 mL reaction system with 50 g / L corn starch.

[0079] Using inositol-3-phosphate synthase D4 as the parent, a modification strategy was designed based on the three-dimensional structural information of the substrate binding pocket. Saturation mutagenesis was performed on amino acid residues D55, N57, Y70, L97, D208, T214, N238, Q243, K257, T260, A273, and I366. First, using the pET28a-D4 recombinant plasmid as a template, degenerate primers were designed using the 22-codon trick for PCR. The PCR reaction was performed using the high-fidelity DNA polymerase KOD One. TMPCR was performed using TOYOBO Master Mix. The PCR reaction system is shown in Table 4.

[0080] Table 4. PCR reaction system

[0081]

[0082] PCR reaction conditions are shown in Table 5:

[0083] Table 5. PCR reaction conditions

[0084]

[0085] The linear DNA products obtained by PCR were subjected to 1% agarose gel electrophoresis. After separation by electrophoresis, the gel was excised and recovered. The nucleic acid concentration of the gel-recovered products was determined using an ultra-micro nucleic acid analyzer, and ligation was performed using a DNA seamless cloning kit (ClonExpress® II One Step Cloning Kit, Novizan). The ligation system is shown in Table 6. The cells were incubated at 37°C for 30 min, and the ligation products were transformed into competent E. coli cells. The transformation process was as described in section 1.2 of Example 1.

[0086] Table 6. PCR product ligation system

[0087]

[0088] Single colonies from the plate were inoculated into 96-well plates containing 300 µL of LB-Kan liquid medium and activated overnight at 37°C and 250 rpm in a shaker. 50 μL of the activated bacterial culture was transferred to a 96-well plate containing 950 μL of TB liquid medium (50 μg / mL kanamycin) and incubated at 37°C and 250 rpm for 2.5 h. The 96-well plates were then incubated on ice for 30 min, and 50 μL of IPTG stock solution (final concentration 0.5 mM) was added to induce expression. The plates were incubated at 22°C and 200 rpm for 20 h. The cells were collected by centrifugation and resuspended in KPi (pH 7.0) buffer to a wet cell concentration of 80 g / L.

[0089] The primary purified enzyme solutions of dextran phosphorylase, glucose phosphate mutase, and inositol monophosphatase were prepared according to the method described in 1.3 of Example 1.

[0090] The components and concentrations in the 2 mL reaction system were as follows: 32 μL / mL of wild-type or mutant bacterial suspension of inositol-3-phosphate synthase prepared as described above; 50 μL / mL of primary purified enzyme solution of dextran phosphorylase; 50 μL / mL of primary purified enzyme solution of glucose phosphate mutase; 50 μL / mL of primary purified enzyme solution of inositol monophosphatase; 100 mM potassium phosphate buffer (pH 7.0); 20 mM magnesium chloride; and 50 g / L corn starch. The 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. The supernatant was then analyzed by LC-MS. The LC-MS detection method and relative activity calculation method were as described above.

[0091] Each site mutation library contained approximately 70 mutants. Analysis showed that mutations at sites T260, K257, N238, T214, and D208 resulted in enzyme inactivation; mutations at sites Y70, L97, and I366 did not significantly alter relative activity; and mutations at sites D55, N57, Q243, and A273, respectively, resulted in some mutants exhibiting significantly increased relative activity. Mutants with increased relative activity were used to repeat the activity experiment as described above, confirming the dominant mutants with significantly increased activity: D4-D55E (aspartic acid at position 55 mutated to glutamic acid), D4-N57S (asparagine at position 57 mutated to serine), and D4-A273R (alanine at position 273 mutated to arginine). Preliminary screening results are attached. Figure 2 .

[0092] Example 4: Obtaining double and triple mutants and their catalytic activity in a 100 mL reaction system with 150 g / L corn starch.

[0093] Based on the initial screening results of site mutations, the following mutants were constructed: D4-D55E, D4-N57S, D4-A273R, D4-D55E / N57S, D4-D55E / N57S / A273T, D4-D55E / N57S / A273P, D4-D55E / N57S / A273N, and D4-D55E / N57S / A273V.

[0094] Following the method described in 1.3 of Example 1, whole-cell enzymes in the form of sludge were prepared, including wild-type and mutant inositol-3-phosphate synthase, dextran phosphorylase, glucose phosphate mutase, and inositol monophosphatase.

[0095] The 100 mL reaction system contained the following components: 25 g / L whole-cell glucan phosphorylase in the form of bacterial sludge, 25 g / L whole-cell glucose-phosphate mutase in the form of bacterial sludge, 10 g / L whole-cell inositol-3-phosphate synthase in the form of bacterial sludge, 25 g / L whole-cell inositol monophosphatase in the form of bacterial sludge, 100 mM potassium phosphate buffer (pH 7.0), 80 mM magnesium chloride, and 150 g / L corn starch. The 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. The supernatant was then analyzed by LC-MS. The LC-MS detection method, inositol content calculation method, and reaction yield calculation method are as described above.

[0096] Finally, the yields of the multi-enzyme combination catalytic reactions of dextran phosphorylase B, glucose phosphate mutase C, inositol monophosphatase E1, and wild-type or different mutant inositol-3-phosphate synthases are shown in Table 7. It can be seen that under the conditions of 100 mL reaction volume, 150 g / L corn starch, and 10 g / L inositol-3-phosphate synthase, the yields of the catalytic reactions of the mutants were significantly improved compared to wild-type D4. Among them, the yield of mutant D4-D55E / N57S / A273P reached 78%, and its amino acid sequence is shown in SEQ ID NO:19.

[0097] Table 7. Comparison of catalytic activities of wild-type inositol-3-phosphate synthase D4 and its mutants in a 100 mL reaction system with 150 g / L corn starch.

[0098] Numbering enzymes Dosage of dextran phosphorylase: 25 g / L Dosage of glucose phosphomutase: 25 g / L Dosage of inositol 3-phosphate synthase: 10 g / L Dosage of inositol monophosphatase: 25g / L Yield % 7-1 B C D4 E1 30 7-2 B C D4-D55E E1 62 7-3 B C D4-N57S E1 51 7-4 B C D4-A273R E1 35 7-5 B C D4-D55E / N57S E1 69 7-6 B C D4-D55E / N57S / A273T E1 72 7-7 B C D4-D55E / N57S / A273P E1 78 7-8 B C D4-D55E / N57S / A273N E1 57 7-9 B C D4-D55E / N57S / A273V E1 65

[0099] Example 5: Results of the reaction catalyzed by the mutant D4-D55E / N57S / A273P and inositol monophosphatase E5 in a 100 mL reaction system with 150 g / L corn starch.

[0100] Following the method described in 1.3 of Example 1, whole-cell enzymes in the form of bacterial sludge were prepared. The 100 mL reaction system contained the following components: 25 g / L whole-cell enzyme of glucan phosphorylase in bacterial sludge form, 25 g / L whole-cell enzyme of glucose-phosphate mutase in bacterial sludge form, 10 g / L whole-cell enzyme of inositol-3-phosphate synthase D4 wild-type or mutant D4-D55E / N57S / A273P in bacterial sludge form, 25 g / L whole-cell enzyme of inositol monophosphatase E5 in bacterial sludge form, 100 mM potassium phosphate buffer (pH 7.0), 80 mM magnesium chloride, and 150 g / L corn starch. The 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. The supernatant was analyzed by LC-MS. The LC-MS detection method, inositol content calculation method, and reaction yield calculation method are as described above.

[0101] The results show that the catalytic yields of the multi-enzyme reaction system composed of isoamylase A, dextran phosphorylase B, glucose phosphate mutase C, inositol monophosphatase E5, and inositol-3-phosphate synthase D4 wild-type and mutant D4-D55E / N57S / A273P were 28% and 75%, respectively, as shown in Table 8. This indicates that the multi-enzyme system composed of mutant D4-D55E / N57S / A273P and inositol monophosphatase E5 also has good catalytic effects for the preparation of inositol.

[0102] Table 8. Comparison of catalytic activities of wild-type inositol 3-phosphate synthase D4 and the combination of mutant D4-D55E / N57S / A273P with inositol monophosphatase E5 in a 100 mL reaction system with 150 g / L corn starch.

[0103] Numbering enzymes Dosage of dextran phosphorylase: 25 g / L Dosage of glucose phosphomutase: 25 g / L Dosage of inositol 3-phosphate synthase: 10 g / L Dosage of inositol monophosphatase: 25 g / L Yield % 8-1 B C D4 E5 28 8-2 B C D4-D55E / N57S / A273P E5 75

[0104] Example 6: Comparison of reaction results with the addition of isoamylase to the catalytic system

[0105] Following the method described in section 1.3 of Example 1, whole-cell enzymes in the form of bacterial sludge were prepared. The 100 mL reaction system contained the following components: 25 g / L whole-cell enzyme of glucan phosphorylase in bacterial sludge form, 25 g / L whole-cell enzyme of glucose-phosphate mutase in bacterial sludge form, 10 g / L whole-cell enzyme of inositol-3-phosphate synthase D4-D55E / N57S / A273P in bacterial sludge form, 25 g / L whole-cell enzyme of inositol monophosphatase E1 in bacterial sludge form, 100 mM potassium phosphate buffer (pH 7.0), 80 mM magnesium chloride, and 150 g / L corn starch. The experimental group received 5 g / L whole-cell enzyme of isoamylase in bacterial sludge form, while the control group received no isoamylase. 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 12,000 rpm for 15 min. The supernatant was then analyzed by LC-MS. The LC-MS detection method, inositol content calculation method, and reaction yield calculation method are as described above.

[0106] The comparison results of the catalytic reaction yields after the addition of isoamylase A are shown in Table 9. The results show that the addition of isoamylase A to the multi-enzyme catalytic system can improve the catalytic efficiency.

[0107] Table 9. Comparison of catalytic reaction results after the addition of isoamylase in a 100 mL reaction system with a substrate concentration of 150 g / L corn starch.

[0108] Numbering enzymes Isoamylase dosage: 5 g / L Dosage of dextran phosphorylase: 25 g / L Dosage of glucose phosphomutase: 25 g / L Dosage of inositol 3-phosphate synthase: 10 g / L Dosage of inositol monophosphatase: 25 g / L Yield % 9-1 / B C D4-D55E / N57S / A273P E1 78 9-2 A B C D4-D55E / N57S / A273P E1 83

[0109] Example 7: Results of the reaction catalyzed by 150 g / L starch, mutant D4-D55E / N57S / A273P, and inositol monophosphatase E1 or E5 in a 1L reaction system.

[0110] Following the method described in 1.3 of Example 1, whole-cell enzymes in the form of bacterial sludge were prepared. The 1L reaction system contained the following components: 5 g / L isoamylase whole-cell enzyme in bacterial sludge form, 25 g / L dextran phosphorylase whole-cell enzyme in bacterial sludge form, 25 g / L glucose phosphate mutase whole-cell enzyme in bacterial sludge form, 10 g / L inositol-3-phosphate synthase whole-cell enzyme D4 wild-type or mutant D4-D55E / N57S / A273P in bacterial sludge form, 25 g / L inositol monophosphatase whole-cell enzyme E1 or E5 in bacterial sludge form, 100 mM potassium phosphate buffer (pH 7.0), 80 mM magnesium chloride, and 150 g / L corn starch. 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. The supernatant was then analyzed by LC-MS. The LC-MS detection method, inositol content calculation method, and reaction yield calculation method are as described above. The detection results are as follows. Figure 3, Figure 4 As shown.

[0111] Ultimately, the catalytic yields of the multi-enzyme reaction system composed of isoamylase A, dextran phosphorylase B, glucose phosphate mutase C, inositol monophosphatase E1, and 10 g / L inositol-3-phosphate synthase D4 wild-type and mutant D4-D55E / N57S / A273P were 35% and 84%, respectively. The catalytic yields of the multi-enzyme reaction system composed of isoamylase A, dextran phosphorylase B, glucose phosphate mutase C, inositol monophosphatase E5, and 10 g / L inositol-3-phosphate synthase D4 wild-type and mutant D4-D55E / N57S / A273P were 32% and 82%, respectively. (See Table 10.)

[0112] Table 10. Results of reactions catalyzed by wild-type inositol 3-phosphate synthase D4 and mutant D4-D55E / N57S / A273P in combination with inositol monophosphatase E1 or E5 in a 1L reaction system with 150 g / L corn starch.

[0113] Numbering enzymes Isoamylase dosage: 5 g / L Dosage of dextran phosphorylase: 25 g / L Dosage of glucose phosphomutase: 25 g / L Dosage of inositol 3-phosphate synthase: 10 g / L Dosage of inositol monophosphatase: 25 g / L Yield % 10-1 A B C D4 E1 35 10-2 A B C D4-D55E / N57S / A273P E1 84 10-3 A B C D4 E5 32 10-4 A B C D4-D55E / N57S / A273P E5 82

[0114] Example 8: Comparison of catalytic dosage between wild-type inositol-3-phosphate synthase D4 and mutant D4-D55E / N57S / A273P under conditions of 1 L reaction volume and 150 g / L starch concentration.

[0115] Following the method described in 1.3 of Example 1, whole-cell enzymes in the form of bacterial sludge were prepared. The 1L reaction system contained the following components: 5 g / L isoamylase whole-cell enzyme in bacterial sludge form, 25 g / L dextran phosphorylase whole-cell enzyme in bacterial sludge form, 25 g / L glucose-phosphate mutase whole-cell enzyme in bacterial sludge form, 64 g / L inositol-3-phosphate synthase D4 whole-cell enzyme or 10 g / L inositol-3-phosphate synthase D4-D55E / N57S / A273P whole-cell enzyme in bacterial sludge form, 25 g / L inositol monophosphatase whole-cell enzyme in bacterial sludge form, 100 mM potassium phosphate buffer (pH 7.0), 80 mM magnesium chloride, and 150 g / L corn starch. 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 12,000 rpm for 15 min. The supernatant was then analyzed by LC-MS. The LC-MS detection method, inositol content calculation method, and reaction yield calculation method are as described above.

[0116] Ultimately, the catalytic yield of the multi-enzyme reaction system composed of isoamylase A, dextran phosphorylase B, glucose phosphate mutase C, inositol monophosphatase E1, and 10 g / L of the D4-D55E / N57S / A273P mutant inositol-3-phosphate synthase was 83.5%, while the catalytic yield using wild-type D4 inositol-3-phosphate synthase at a concentration of 64 g / L was 85%. This indicates that by directed evolutionary modification of wild-type inositol-3-phosphate synthase D4, the catalytic efficiency of the mutant D4-D55E / N57S / A273P is significantly improved, which can effectively reduce the amount of enzyme used in the catalytic synthesis of inositol. The results are shown in Table 11.

[0117] Table 11. Comparison of catalytic dosage between wild-type inositol-3-phosphate synthase D4 and mutant D4-D55E / N57S / A273P in a 1L reaction system with 150 g / L corn starch.

[0118] Numbering enzymes Isoamylase dosage: 5 g / L Dosage of dextran phosphorylase: 25 g / L Dosage of glucose phosphomutase: 25 g / L Inositol 3-phosphate synthase Dosage of inositol monophosphatase: 25 g / L Yield % 11-1 A B C D4-Wild Type Dosage: 64 g / L E1 85 11-2 A B C Dosage of D4-D55E / N57S / A273P: 10 g / L E1 83.5

Claims

1. A mutant of inositol-3-phosphate synthase, characterized in that: It uses inositol-3-phosphate synthase, whose amino acid sequence is shown in SEQ ID NO:4, as the parent, and mutates aspartic acid at position 55 to glutamic acid to obtain mutant D4-D55E.

2. A mutant of inositol-3-phosphate synthase, characterized in that: It uses the mutant D4-D55E described in claim 1 as the parent, and mutates asparagine to serine at position 57 to obtain mutant D4-D55E / N57S.

3. A mutant of inositol-3-phosphate synthase, characterized in that: It uses the mutant D4-D55E / N57S described in claim 2 as the parent, and mutates alanine at position 273 to threonine, proline, asparagine, and valine, respectively, to obtain mutants: D4-D55E / N57S / A273T, D4-D55E / N57S / A273P, D4-D55E / N57S / A273N, and D4-D55E / N57S / A273V.

4. The use of the inositol-3-phosphate synthase mutant according to any one of claims 1-3 in the synthesis of inositol.

5. A method for preparing inositol, characterized in that: It uses starch as a substrate and adds glucan phosphorylase, glucose phosphate mutase, the inositol-3-phosphate synthase mutant according to any one of claims 1-3, and inositol monophosphatase as enzyme catalysts to catalyze the production of inositol from the substrate; wherein the inositol monophosphatase is derived from Archaeoglobusfulgidus, and the amino acid sequence is shown in SEQ ID NO:5; or, the inositol monophosphatase is derived from Thermotoganeapolitana, and the amino acid sequence is shown in SEQ ID NO:

8.

6. The method for preparing inositol according to claim 5, characterized in that: The enzyme catalyst also includes isoamylase.

7. The method for preparing inositol according to claim 5, characterized in that: It includes the following steps: a. Weigh out starch and add glucan phosphorylase, glucose phosphorylation mutase, inositol-3-phosphate synthase mutant, and inositol monophosphatase. b. Perform the enzyme-catalyzed reaction at 60-90℃ for 10-120 hours to obtain the product.

8. The method for preparing inositol according to claim 7, characterized in that: The amount of starch used in step a in the reaction system is 10 g / L to 150 g / L; The enzyme-catalyzed reaction in step b is carried out at a temperature of 80°C for 20 hours.

9. The method for preparing inositol according to any one of claims 5-7, characterized in that: The inositol-3-phosphate synthase mutant and inositol monophosphatase are in the form of primary purified enzyme solution, homogenized enzyme solution or whole cell enzyme.

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