Myo-inositol-3-phosphate synthase mutants and their use in the production of myo-inositol

Abstract

This invention belongs to the field of genetic engineering technology and discloses a mutant of inositol-3-phosphate synthase and its application in inositol preparation. This invention involves site-directed mutagenesis of the inositol-3-phosphate synthase shown in SEQ ID NO.1, mutating valine at position 139 to isoleucine, resulting in a mutant with significantly enhanced enzyme activity. This mutant exhibits a specific activity of 1.39 U / mg, representing a 69% increase in enzyme activity compared to the parental inositol-3-phosphate synthase Mt-IPS. When used in inositol preparation, it synergistically enhances the conversion of glucose to inositol in a three-enzyme two-step cascade reaction system.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to inositol-3-phosphate synthase mutants and their application in inositol preparation. Background Technology

[0002] Inositol, also known as cyclohexanehexol, is an indispensable growth factor widely distributed in nature. It can be detected in everything from soil bacteria (such as *Escherichia coli* and *Bacillus subtilis*), fungi commonly used in fermentation industries (such as *Saccharomyces cerevisiae* and *Aspergillus niger*), to mammalian tissues and organs (especially metabolically active sites such as the brain, liver, and kidneys), and even in the seeds, fruits, and leaves of higher plants. In living organisms, inositol is not only a key component of cell membrane phospholipids (such as phosphatidylinositol), maintaining membrane fluidity and stability, but also the core structural basis of many secondary messenger molecules in eukaryotic cells (such as inositol triphosphate 3P-I and phosphatidylinositol 4,5-bisphosphate). It deeply participates in various important cellular physiological processes, playing a crucial role in cell growth, survival, development, nerve function, skeletal function, and reproduction.

[0003] Currently, inositol has been widely used in multiple industries. In the food industry, it is often added as a nutritional fortifier to dairy products, cereal products, and functional beverages to supplement essential nutrients needed by the human body. In animal feed, it is frequently used to regulate animal metabolism and promote growth and development. In the pharmaceutical industry, due to its lipid metabolism-promoting effects, inositol has been directly formulated into tablets for the treatment of chronic interstitial hepatitis, fatty liver, cirrhosis, and other diseases. It also possesses insulin-mimicking activity, effectively lowering blood sugar levels, making it valuable in diabetes management. Furthermore, inositol naturally exhibits moderate anticancer activity: a pilot clinical trial showed that when used in combination with inositol hexaphosphate, it enhanced the anticancer effects of conventional chemotherapy and slowed the progression of cancer metastasis. Moreover, recent literature indicates that inositol shows promising therapeutic potential in the intervention of depression, Alzheimer's disease, panic disorder, obsessive-compulsive disorder, autism, post-traumatic stress disorder, and pain control.

[0004] Existing methods for synthesizing inositol mainly include chemical synthesis, microbial synthesis, and enzymatic synthesis. While chemical synthesis utilizes abundant raw materials and mature technology, it suffers from high production costs and severe pollution. Microbial synthesis, using the enzymatic reaction system of microorganisms, offers advantages such as low cost, low pollution, and easy product separation, but suffers from long fermentation cycles and low production efficiency. Enzymatic synthesis, using glucose, glucose-6-phosphate, and other substrates, introduces multiple key enzymes into an in vitro reaction system to catalyze the production of inositol. This method boasts advantages such as high product purity, high reaction efficiency, simple reaction system, and strong controllability, making it highly promising for large-scale industrial production of inositol.

[0005] Inositol-3-phosphate synthase is a key rate-limiting enzyme in the inositol biosynthesis pathway. It catalyzes the conversion of glucose-6-phosphate to inositol-3-phosphate, which is the first crucial step in inositol synthesis and directly determines the synthesis efficiency and yield. However, naturally occurring inositol-3-phosphate synthase has limited catalytic activity and is easily inactivated in in vitro reaction systems, making it difficult to meet the industrial production requirements for enzymes with high catalytic efficiency and high stability. This severely restricts the industrialization process of inositol synthesis via enzymatic catalysis.

[0006] In view of this, this invention is hereby proposed. Summary of the Invention

[0007] Based on the above problems, the purpose of this invention is to provide an inositol-3-phosphate synthase mutant and its application in the preparation of inositol. The mutant has significantly improved enzyme activity and can catalyze the reaction more efficiently.

[0008] The first aspect of the present invention provides an inositol-3-phosphate synthase mutant, which is based on the inositol-3-phosphate synthase shown in SEQ ID NO.1 by mutating valine at position 139 to isoleucine.

[0009] A second aspect of the invention provides a gene encoding the above-mentioned inositol-3-phosphate synthase mutant.

[0010] A third aspect of the present invention provides a recombinant expression vector comprising the above-described gene.

[0011] Furthermore, the vector for the recombinant expression vector is plasmid pHT43.

[0012] A fourth aspect of the present invention provides a recombinant strain comprising the above-described recombinant expression vector.

[0013] Furthermore, the host cell of the recombinant strain is... B. subtilis SCK6.

[0014] The fifth aspect of the present invention provides the application of the above-mentioned inositol-3-phosphate synthase mutant in the preparation of inositol.

[0015] The sixth aspect of the present invention provides a method for preparing inositol, comprising: firstly, using glucophosphokinase to perform a first-step catalytic reaction on glucose and sodium hexametaphosphate, and after the glucose is completely converted into glucose-6-phosphate, adding the inositol-3-phosphate synthase mutant and inositol monophosphatase as described in claim 1 to perform a second-step catalytic reaction.

[0016] Furthermore, the concentration ratio of glucose to sodium hexametaphosphate is 2:1 to 4:1.

[0017] Furthermore, the pH value of the system for the second step catalytic reaction is 7.0-7.5.

[0018] Compared with the prior art, the present invention has the following beneficial effects: 1. This invention uses molecular docking technology to pinpoint the core amino acid active site of inositol-3-phosphate synthase Mt-IPS, providing a clear target for enzyme modification. Subsequently, site-directed mutagenesis was used to mutate inositol-3-phosphate synthase Mt-IPS, obtaining a mutant inositol-3-phosphate synthase with higher enzyme activity. This mutant exhibits a specific activity of 1.39 U / mg, representing a 69% increase in enzyme activity compared to the parental inositol-3-phosphate synthase Mt-IPS.

[0019] 2. This invention avoids the inhibitory effect of sodium hexametaphosphate on inositol-3-phosphate synthase by establishing a three-enzyme two-step cascade reaction system, thus achieving high conversion of glucose to inositol. Attached Figure Description

[0020] Figure 1 A three-dimensional structural diagram of Mt-IPS; Figure 2 The figure shows the simulation results of molecular docking between Mt-IPS and glucose-6-phosphate. The numbers 2.1, 2.2, etc. in the figure represent the hydrogen bond lengths at the protein-small molecule connection sites. Detailed Implementation

[0021] The invention is further described below through specific embodiments. Unless otherwise specified, the technical means and materials involved in the following embodiments are all known to those skilled in the art, and suitable means and materials that can solve the corresponding technical problems can be selected. In addition, the embodiments should be understood as illustrative, not limiting the scope of the invention, and the essence and scope of the invention are defined only by the claims.

[0022] It should be understood that the scope of this invention is not limited to the defined processes, properties, or components, as these embodiments and other descriptions are merely illustrative of specific aspects of the invention. In fact, various modifications to these embodiments that will be apparent to those skilled in the art or related fields without departing from the spirit and scope of this invention are covered within the scope of the appended claims.

[0023] It should be noted that, unless otherwise defined, the scientific and technical terms used in the context of this invention should have the meanings commonly understood by those skilled in the art.

[0024] The markers for the inositol-3-phosphate synthase mutant used in this invention are as follows: The term "amino acid with original amino acid position substitution" is used to represent the mutated amino acid of inositol-3-phosphate synthase. For example, K138R indicates that the amino acid at position 138 is replaced by R from the parental inositol-3-phosphate synthase, and the position number corresponds to the amino acid sequence number in SEQ ID NO.1.

[0025] This invention provides a mutant of inositol-3-phosphate synthase, characterized in that the mutant is based on the inositol-3-phosphate synthase shown in SEQ ID NO.1, by mutating valine at position 139 to isoleucine.

[0026] This invention uses inositol-3-phosphate synthase Mt-IPS, whose amino acid sequence is shown in SEQ ID NO.1, as the parent. First, by analyzing the molecular docking results between Mt-IPS and glucose-6-phosphate, it was determined that lysine at position 138 (LYS-138), valine at position 139 (VAL-139), alanine at position 164 (ALA-164), and lysine at position 329 (LYS-329) are possible key binding sites in the specific interaction between Mt-IPS and the substrate glucose-6-phosphate. Among them, the positively charged side chain of lysine is very likely to form a stable bond with the negatively charged phosphate group in the glucose-6-phosphate molecule through electrostatic attraction. It may also act as a hydrogen bond donor to form hydrogen bonds with oxygen atoms in the substrate molecule, thereby enhancing the affinity between the enzyme and the substrate. As a hydrophobic amino acid, valine's side chain may be inserted into the hydrophobic region of the glucose-6-phosphate molecule through hydrophobic interactions, further stabilizing the structure of the enzyme-substrate complex. Although alanine has a small side chain, it may provide a suitable spatial environment for substrate binding through steric hindrance or synergistic effects with surrounding amino acid residues, ensuring that the substrate can be accurately inserted into the active site of the enzyme.

[0027] Furthermore, the present invention performs site-directed mutagenesis on the above-mentioned sites, constructs a mutant library, and measures the enzyme activity of the mutants, ultimately screening out mutants of inositol-3-phosphate synthase with significantly enhanced catalytic activity.

[0028] K138R: Both LYS and ARG carry a positive charge. However, ARG has a longer side chain and can form more hydrogen bonds or electrostatic interactions. Mutating LYS-138 to ARG may increase the binding force with negatively charged substrates or ligands, stabilize the substrate-enzyme complex, and thus improve the rate of enzyme-catalyzed reaction.

[0029] V139I: Both VAL and ILE belong to nonpolar aliphatic amino acids, but isoleucine has a larger and more branched side chain than valine. This may optimize the hydrophobic environment of the enzyme's active site, enhance the binding ability to hydrophobic substrates or ligands, and make the substrate bind more stably to the active site, thereby improving catalytic efficiency and enhancing enzyme activity.

[0030] A164P: ALA has a smaller side chain, while PRO has a unique cyclic structure. When ALA-164 mutates to PRO, it may cause a fine-tuning of the local protein conformation, making the spatial structure of the active site more compatible with the substrate, promoting substrate binding and catalytic reactions, and thus enhancing enzyme activity.

[0031] A164Y: ALA has a simple side chain, while TYR has a phenolic hydroxyl group on its side chain. When ALA-164 is mutated to TYR, the introduced phenolic hydroxyl group can participate in the formation of new hydrogen bonds or other weak interactions, increasing the interaction between the enzyme and the substrate. This helps the substrate to be correctly positioned and stabilized at the active site, thereby improving the enzyme's activity.

[0032] K329H: LYS mainly participates in electrostatic interactions, while HIS has special acid-base properties under physiological pH conditions and can participate in acid-base catalysis as a proton donor or acceptor. If LYS-329 involves key steps such as proton transfer in catalysis, mutation into HIS may optimize the catalytic mechanism, accelerate the reaction process, and thus enhance enzyme activity.

[0033] The present invention further provides a gene encoding the inositol-3-phosphate synthase mutant as described above.

[0034] Embodiments of the present invention further provide a recombinant expression vector containing the genes described above. In some preferred embodiments, the vector for the recombinant expression vector is plasmid pHT43.

[0035] Embodiments of the present invention further provide a recombinant bacterial strain comprising the recombinant expression vector described above. In some preferred embodiments, the host cell of the recombinant bacterial strain is... B. subtilis SCK6.

[0036] This invention further provides the application of the inositol-3-phosphate synthase mutant in the preparation of inositol. Using the inositol-3-phosphate synthase mutant of this embodiment, the conversion of glucose to inositol is significantly improved compared to the parental inositol-3-phosphate synthase.

[0037] The present invention further provides a method for preparing inositol, comprising: firstly, using glucophosphokinase to perform a first-step catalytic reaction on glucose and sodium hexametaphosphate, and after the glucose is completely converted into glucose-6-phosphate, adding the inositol-3-phosphate synthase mutant and inositol monophosphatase as described in claim 1 to perform a second-step catalytic reaction.

[0038] It should be noted that existing enzyme-catalyzed preparations of inositol mostly employ a "one-pot" method, where multiple enzymes are directly mixed with the substrate for the reaction. However, regarding the novel enzyme cascade reaction system constructed in this embodiment, which includes the aforementioned three enzymes and the phosphate donor sodium hexametaphosphate, the applicant's long-term research has revealed that sodium hexametaphosphate significantly inhibits inositol-3-phosphate synthase, leading to low inositol synthesis efficiency and poor yield. Therefore, this embodiment constructs a three-enzyme two-step cascade reaction system, strictly controlling the glucose to sodium hexametaphosphate concentration ratio in the reaction system to be 2:1-4:1. Simultaneously, the pH of the second-step catalytic reaction is adjusted to 7.0-7.5. This ensures efficient supply and full utilization of phosphate groups during the reaction process, effectively avoiding the inhibitory effect of sodium hexametaphosphate on inositol-3-phosphate synthase, and maintaining a suitable pH environment for the key enzyme in the second step reaction. Ultimately, this significantly improves the catalytic conversion efficiency of inositol, achieving high-yield preparation of inositol.

[0039] In this specific embodiment, the first step reaction is carried out in a 50 mM Tris-HCl buffer at pH 7.0. The reaction system also includes: 10 U / mL glucokinase, 50 mM glucose, 12.5-25 mM sodium hexametaphosphate, 5 mM MgCl2, and 5 mM NH4Cl. Since the pH of the reaction system changes due to the accumulation of metabolites during the reaction, affecting enzyme activity, after the first step reaction, the pH of the reaction system is readjusted to the range of 7.0-7.5 using 1M NaOH. Then, 5 U / mL inositol-3-phosphate synthase mutant and 5 U / mL inositol monophosphatase are added to carry out the second step reaction. During the second step reaction, the pH of the system continues to decrease. Therefore, every hour, the pH needs to be readjusted to the range of 7.0-7.5 using 1M NaOH to maintain the optimal pH environment for the enzyme, ultimately achieving a high yield of inositol. The second step reaction takes 24 hours, and all reactions are carried out under constant temperature water bath conditions at 37°C.

[0040] To make the technical solution of the present invention clearer, the following detailed description of the inositol-3-phosphate synthase mutant is provided through several specific embodiments.

[0041] The experimental reagents involved in the embodiments of this invention include: LB liquid medium: trypsin 10.0 g / L, sodium chloride 5.0 g / L, yeast extract 10.0 g / L, chloramphenicol 25 μg / mL, erythromycin 1 μg / mL, pH 7.2.

[0042] LB solid medium: trypsin 10.0 g / L, sodium chloride 5.0 g / L, yeast extract 10.0 g / L, agar 15 g / L, chloramphenicol 25 μg / mL, erythromycin 1 μg / mL, pH 7.2.

[0043] High-fidelity enzyme mixture, T4 DNA ligase, and primers were all purchased from Shanghai Sangon Biotech Co., Ltd. Gel purification kit: purchased from Tiangen Biotech (Beijing) Co., Ltd.; All other chemical reagents were purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd.

[0044] The indicators and their measurement methods involved in the embodiments of this invention include: Inositol-3-phosphate synthase (IPS) activity was measured at 37°C in a 50 mM Tris-HCl buffer (pH 7.0) containing 50 mM glucose-6-phosphate (a specific substrate for IPS), 5 mM NH4Cl (as an activator to promote catalytic conformation formation of the enzyme), and excess inositol monophosphatase (used for the coupling reaction to hydrolyze inositol-3-phosphate generated by IPS to inositol and inorganic phosphate). After the reaction was initiated, IPS catalyzed the cyclization of glucose-6-phosphate to inositol-3-phosphate, followed by rapid hydrolysis of inositol-3-phosphate by inositol monophosphatase to release inorganic phosphate. The amount of inorganic phosphate generated was quantitatively detected using a mild pH phosphate assay, thus reflecting the catalytic activity of IPS.

[0045] Enzyme activity units are defined as the amount of enzyme required to catalyze the production of 1 micromole of product per minute under the specific reaction conditions described above, expressed in units of U. Specific enzyme activity refers to the number of enzyme activity units per milligram of protein under the specific reaction conditions described above, expressed in units of U / mg.

[0046] Example 1: Construction, expression, and purification of inositol-3-phosphate synthase mutants (1) Obtain the mutant gene of inositol-3-phosphate synthase This invention first tested the enzyme activity of inositol-3-phosphate synthase from five different microbial sources, including *Mycobacterium tuberculosis*, *Kluyveromyces martensii*, *Hansenula polymorpha*, and *Cyclophorus delavayi*. The enzyme activity test showed that the inositol-3-phosphate synthase Mt-IPS (amino acid sequence shown in SEQ ID NO.1) from *Mycobacterium tuberculosis* exhibited the highest enzyme activity, with a specific activity of 0.82 U / mg.

[0047] Figure 1 The figure shows the three-dimensional structure of Mt-IPS. This enzyme is a protein composed of 365 amino acids linked by peptide bonds. Its spatial structure contains a variety of characteristic secondary structural elements: the red-marked parts are α-helices, a common protein secondary structure where amino acid residues form a right-handed helical conformation along the helical axis through hydrogen bonds, similar to a helical spring, providing a certain degree of rigidity and stability to the enzyme molecule; the yellow-marked parts are β-sheets, composed of several almost fully extended polypeptide chains laterally linked by hydrogen bonds, forming a layered structure, like stacked pages of a book, which is an important support for maintaining the spatial conformation of the enzyme molecule; the green-marked parts are random coils, which do not have a fixed periodic conformation and have relatively flexible mobility, playing a key regulatory role in enzyme-substrate binding, conformational changes, and catalysis. These different secondary structures, through a specific spatial arrangement, together constitute the overall three-dimensional structure of Mt-IPS, and this structure is closely related to the enzyme's catalytic function, substrate specificity, and stability.

[0048] Based on the three-dimensional structure of Mt-IPS, this invention further simulates its molecular docking with glucose-6-phosphate, and the results are as follows: Figure 2 As shown, lysine at position 135 (LYS-135), lysine at position 138 (LYS-138), valine at position 139 (VAL-139), alanine at position 164 (ALA-164), and lysine at position 329 (LYS-329) are potential key binding sites in the specific interaction between Mt-IPS and the substrate glucose-6-phosphate.

[0049] Furthermore, in order to verify the actual role of the above-mentioned predicted sites in the function of the enzyme and to explore their specific effects on the activity of Mt-IPS enzyme, this invention selected some amino acid active sites for site-directed mutagenesis. The mutagenesis sites include: K138R, V139I, K329H, A164P, and A164Y.

[0050] To achieve site-directed mutagenesis at specific amino acid sites, primer design follows the principle of overlap extension PCR, and two pairs of primers are constructed using the following strategy: The first pair consists of specific primers (F1 and R1) containing mutation sites: the forward primer F1 needs to introduce a base change at the target mutation site, while ensuring that 15 bases matching the template are retained on each side of the mutation site to guarantee annealing specificity; the reverse primer R1 and F1 form a complementary overlap in the mutation region (overlap length 15 bp), and its sequence corresponds to the reverse complementary strand after mutation, which also contains the complementary bases of the mutation site.

[0051] The second pair consists of universal vector primers (F2 and R2): F2 binds to the vector sequence upstream of the target gene, and R2 binds to the vector sequence downstream of the target gene. Both are located outside the mutation region, and the amplification range must cover the entire target gene and mutation site to ensure that the second round of PCR can splice the complete mutant gene.

[0052] The experimental procedure consisted of two rounds of PCR: In the first round, using the original recombinant plasmid as a template, amplification was performed using F1+R2 and F2+R1 respectively, yielding two DNA fragments containing mutations. The F1+R2 amplification product contained the upstream sequence and the mutated region, while the F2+R1 amplification product contained the mutated region and the downstream sequence. The two products had overlapping regions within the mutated regions. In the second round of PCR, equal amounts of the amplification products from both rounds were mixed as a template (no purification required). Utilizing the complementary pairing of the overlapping regions of the two products, extension and splicing were performed under the guidance of F2 and R2 to obtain the full-length mutated gene. The PCR reaction system is shown in Table 1, the reaction procedure in Table 2, and the primers used in Table 3.

[0053] Table 1. Mt-IPS site-directed mutagenesis PCR reaction system .

[0054] Table 2 Mt-IPS site-directed mutagenesis PCR reaction procedure .

[0055] Table 3 Mt-IPS site-directed mutagenesis primers .

[0056] Through site-directed mutagenesis, the following mutant genes of inositol-3-phosphate synthase were obtained: Mt-IPS-K138R, Mt-IPS-V139I, Mt-IPS-K329H, Mt-IPS-A164P, and Mt-IPS-A164Y.

[0057] (2) Construction of plasmid multimers The inositol-3-phosphate synthase mutant gene and the Pht43 plasmid were amplified separately. For the Pht43 plasmid, specific primers were used for PCR. The designed primer sequences accurately identified the target regions at both ends of the plasmid. Under the catalysis of thermostable DNA polymerase, the plasmid was linearized and amplified, ensuring that the obtained linearized plasmid fragments had clear sequence characteristics at both ends. For the mutant gene, specific primers were also designed based on its sequence. Genomic DNA containing the mutant gene was used as a template for amplification, so that the mutant gene fragments formed specific sequence regions complementary to the ends of the linearized plasmid, laying the foundation for the subsequent ligation reaction. The PCR amplification reaction system for the Pht43 plasmid and the inositol-3-phosphate synthase mutant gene is the same as in Table 1, the amplification reaction procedure is the same as in Table 2, and the primers used are listed in Table 4.

[0058] Table 4 Primers for PCR amplification of Pht43 plasmid and inositol-3-phosphate synthase mutant gene .

[0059] After amplification, the linearized Pht43 plasmid and the mutant gene fragment are ready to serve as primers and templates for each other. In the subsequent PCR reaction, during the annealing stage, due to the presence of identical base sequence regions at their ends, the single-stranded linearized plasmid and the single-stranded mutant gene fragment bind specifically to these identical regions through base pairing, forming a nicked hybridization intermediate. In the extension step, DNA polymerase uses this hybridization intermediate as a template and, according to base pairing, utilizes free deoxyribonucleotides in the reaction system to extend from the binding region towards both ends, filling the gap and continuously elongating the strand. After multiple PCR cycles, the linearized plasmid and mutant gene fragment alternately ligate, ultimately yielding a large number of amplified plasmid multimers with a repeating pattern of mutant gene-plasmid gene-mutant gene-… The specific PCR reaction system is shown in Table 5, and the reaction procedure is shown in Table 6. The following inositol-3-phosphate synthase mutant plasmids were obtained: Pht43-Mt-IPS-K138R, Pht43-Mt-IPS-V139I, Pht43-Mt-IPS-K329H, Pht43-Mt-IPS-A164P, and Pht43-Mt-IPS-A164Y.

[0060] Table 5 Reaction system for constructing plasmid multimers .

[0061] Table 6 Reaction procedures for constructing plasmid multimers .

[0062] (3) Construction of recombinant engineered bacteria Take from laboratory collection B. subtilis SCK6 glycerol bacteria were revived and activated using LB solid plates containing 1 μg / mL erythromycin. Single colonies with uniform morphology and neat edges were picked from the plates and inoculated into 100 mL of LB liquid medium. The culture was carried out overnight in a shaker at 37°C and 220 rpm.

[0063] The following day, fresh bacterial culture was mixed with an equal volume of LB liquid medium containing 10 mg / mL xylose, and the OD of the bacterial culture was adjusted. 600 To obtain approximately 1, the adjusted bacterial culture was incubated at 37°C and 220 rpm for 2 hours. B. subtilis SCK6 competent cell sap.

[0064] Take 100 μL of freshly prepared competent cell culture and place it in a sterile 1.5 mL EP tube. Add 1 μL of the above plasmid polymer (concentration approximately 50-100 ng / μL) and gently mix with a sterile pipette tip. Incubate the EP tube at 37°C and 220 rpm for 90 min.

[0065] After resuscitation, all cell suspension was evenly spread onto LB solid selective plates containing 25 μg / mL chloramphenicol and 1 μg / mL erythromycin. After complete absorption by the bacterial suspension, the plates were inverted and sealed, and incubated at 37°C for 16 h. Positive colonies were picked for PCR sequencing identification to obtain recombinant engineered bacteria.

[0066] (4) Cell culture and enzyme purification Single colonies of the recombinant engineered bacteria, whose transformation was confirmed by PCR and sequencing, were inoculated into LB liquid medium containing 25 μg / mL chloramphenicol and 1 μg / mL erythromycin. The culture was then placed in a shaker at 37°C and 220 rpm for 12 h. Subsequently, 50 mL of the bacterial culture was placed in a centrifuge tube and centrifuged at 8000 rpm for 10 min at 4°C. The supernatant was discarded and the bacterial pellet was collected.

[0067] Add 10 mL of pre-cooled buffer (50 mM Tris-HCl containing 300 mM sodium chloride, pH 8.0) to the bacterial pellet, and repeatedly pipette to fully resuspend the cells. Lyse the cells using an ultrasonic cell disruptor under ice bath conditions. After lysis, centrifuge the bacterial solution at 4°C and 12,000 rpm for 20 min, and transfer the supernatant (containing soluble recombinant protein) to a new centrifuge tube. Filter the solution through a 0.22 μm filter membrane to remove impurities.

[0068] Recombinant protein purification was performed using a nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography column: the column was first washed with equilibration buffer (50 mM Tris-HCl containing 300 mM sodium chloride, pH 8.0) until the baseline stabilized. The filtered supernatant was then loaded onto the column. After complete adsorption of the sample, the column was washed with 50 mM Tris-HCl (pH 8.0) containing 300 mM sodium chloride and 50 mM imidazole. The target protein was then eluted with 50 mM Tris-HCl (pH 8.0) containing 300 mM sodium chloride and 300 mM imidazole. The elution fraction was collected in 2 mL increments per tube.

[0069] The collected active components were placed in dialysis bags (molecular weight cutoff 10 kDa) and in 50 mM Tris-HCl buffer (pH 8.0). Dialysis was performed at 4°C for 24 h, with the buffer changed multiple times to thoroughly remove imidazole. The dialyzed protein solution was then subjected to SDS-PAGE analysis: a 12% separating gel and a 5% stacking gel were prepared. 20 μg of protein sample was loaded and electrophoresed at a constant voltage of 80 V until bromophenol blue entered the separating gel. The voltage was adjusted to 120 V and electrophoresis continued until the indicator reached the bottom of the gel. After Coomassie brilliant blue staining for 2 h and destaining with destaining solution until the bands were clear, the purity of the recombinant protein was confirmed by observing whether a single protein band appeared and the consistency of the band molecular weight with the theoretical value. The following five inositol-3-phosphate synthase mutants were obtained: Mt-IPS-K138R, Mt-IPS-V139I, Mt-IPS-K329H, Mt-IPS-A164P, and Mt-IPS-A164Y. Enzyme activity assays were performed on the obtained inositol-3-phosphate synthase mutants. The results showed that only the mutant Mt-IPS-V139I had higher enzyme activity than the parent Mt-IPS, with a specific activity of 1.39 U / mg, which was 69% higher than that of Mt-IPS.

[0070] The obtained inositol-3-phosphate synthase mutant Mt-IPS-V139I was sequenced, and the amino acid sequence is shown in SEQ ID NO.2.

[0071] Example 2: Application of the inositol-3-phosphate synthase mutant Mt-IPS-V139I in inositol preparation The above-mentioned inositol-3-phosphate synthase mutant Mt-IPS-V139I was used to prepare inositol. The preparation method is as follows: Add 10 U / mL glucokinase, 50 mM glucose, 12.5 mM sodium hexametaphosphate, 5 mM MgCl2 and 5 mM NH4Cl to 50 mM Tris-HCl buffer at pH 7.0. Perform the first step reaction in a constant temperature water bath at 37 ℃. After glucose is completely converted to glucose-6-phosphate, readjust the pH of the reaction system to 7.0 using 1M NaOH. Then add 5 U / mL inositol-3-phosphate synthase mutant and 5 U / mL inositol monophosphatase. Perform the second step reaction in a constant temperature water bath at 37 ℃ for 24 h.

[0072] In the above preparation methods, the polyphosphokinase is derived from Mycobacterium caspaviae ( Mycobacterium canettii Its amino acid sequence is shown in SEQ ID NO.3, and its specific activity is 46.3 U / mg; inositol monophosphatase is derived from Escherichia coli (E. coli). E. coli Its amino acid sequence is shown in SEQ ID NO.4, and its specific activity is 93.5 U / mg.

[0073] The parental inositol-3-phosphate synthase Mt-IPS was used as a control, that is, the inositol-3-phosphate synthase mutant Mt-IPS-V139I in the above preparation method was replaced with Mt-IPS.

[0074] The concentration and conversion rate of inositol in the two reaction systems were determined by high performance liquid chromatography. The inositol concentration in the reaction system using Mt-IPS-V139I was 45.2 mM and the conversion rate was 90.4%; the inositol concentration in the reaction system using Mt-IPS was 34.4 mM and the conversion rate was 68.8%.

[0075] Finally, it should be noted that although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A mutant of inositol-3-phosphate synthase, characterized in that, The mutant is based on the inositol-3-phosphate synthase with the amino acid sequence shown in SEQ ID NO.1, and the valine at position 139 is mutated to isoleucine.

2. The gene encoding the inositol-3-phosphate synthase mutant as described in claim 1.

3. A recombinant expression vector, characterized in that, It contains the gene as described in claim 2.

4. The recombinant expression vector as described in claim 3, characterized in that, The recombinant expression vector is carried by plasmid pHT43.

5. A recombinant strain, characterized in that, It includes the recombinant expression vector as described in claim 3 or 4.

6. The recombinant strain according to claim 5, characterized in that, The host cell of the recombinant strain is B. subtilis SCK6.

7. The application of the inositol-3-phosphate synthase mutant as described in claim 1 in the preparation of inositol.

8. A method for preparing inositol, characterized in that, include: First, glucose and sodium hexametaphosphate are catalyzed using glucophosphokinase. Once the glucose is completely converted to glucose-6-phosphate, the inositol-3-phosphate synthase mutant and inositol monophosphatase described in claim 1 are added to carry out the second catalytic reaction.

9. The preparation method according to claim 8, characterized in that, The concentration ratio of glucose to sodium hexametaphosphate is 2:1 to 4:

1.

10. The preparation method according to claim 8, characterized in that, The pH value of the system for the second step of the catalytic reaction is 7.0-7.5.

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